Cartilage and bone repair and regeneration using postpartum-derived cells

ABSTRACT

Cells derived from postpartum tissue and methods for their isolation and induction to differentiate to cells of a chondrogenic or osteogenic phenotype are provided by the invention. The invention further provides cultures and compositions of the postpartum-derived cells and products related thereto. The postpartum-derived cells of the invention and products related thereto have a plethora of uses, including but not limited to research, diagnostic, and therapeutic applications, for example, in the treatment of bone and cartilage conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit of U.S. Provisional Application Ser. No. 60/483,264,filed Jun. 27, 2003, the entire contents of which are incorporated byreference herein. Other related applications include the followingcommonly-owned, co-pending applications, the entire contents of each ofwhich are incorporated by reference herein: U.S. application No.[ETH-5073 NP1], filed Jun. 25, 2004, U.S. application No. [ETH-5073NP2], filed Jun. 25, 2004, U.S. application No. [ETH-5073 NP3], filedJun. 25, 2004, U.S. application No. [ETH-5073 NP4], filed Jun. 25, 2004,U.S. application No. [ETH-5073 NP5], filed Jun. 25, 2004, U.S.application No. [ETH-5073 NP7], filed Jun. 25, 2004, and U.S.Provisional Application No. 60/555,908 [ETH 5127], filed Mar. 24, 2004.[ETH 5127], filed Mar. 24, 2004.

FIELD OF THE INVENTION

This invention relates to the field of mammalian cell biology and cellculture. In particular, the invention relates to cells derived frompostpartum tissue having the potential to differentiate intochondrogenic and osteogenic lineages, and methods of preparation and useof those postpartum tissue-derived cells, including cell-based therapiesfor conditions of bone and cartilage.

BACKGROUND OF THE INVENTION

Diseases and conditions of bone and cartilage affect a large portion ofthe population. Three types of cartilage are present in mammals andinclude: hyaline cartilage, fibrocartilage, and elastic cartilage.Hyaline cartilage consists of a gristly mass having a firm, elasticconsistency, is translucent and pearly blue in color. Hyaline cartilageis predominantly found on the articulating surfaces of articulatingjoints. It is found also in epiphyseal plates, costal cartilage,tracheal cartilage, bronchial cartilage and nasal cartilage.Fibrocartilage is essentially the same as hyaline cartilage except thatit contains fibrils of type I collagen that add tensile strength to thecartilage. The collagenous fibers are arranged in bundles, with thecartilage cells located between the bundles. Fibrocartilage is foundcommonly in the annulus fibrosis of the invertebral disc, tendonous andligamentous insertions, menisci, the symphysis pubis, and insertions ofjoint capsules. Elastic cartilage also is similar to hyaline cartilageexcept that it contains fibers of elastin. It is more opaque thanhyaline cartilage and is more flexible and pliant. These characteristicsare defined in part by the elastic fibers embedded in the cartilagematrix. Typically, elastic cartilage is present in the pinna of theears, the epiglottis, and the larynx.

The surfaces of articulating bones in mammalian joints are covered witharticular cartilage. The articular cartilage prevents direct contact ofthe opposing bone surfaces and permits the near frictionless movement ofthe articulating bones relative to one another. Two types of articularcartilage defects are commonly observed in mammals and includefull-thickness and partial-thickness defects. The two types of defectsdiffer not only in the extent of physical damage but also in the natureof repair response each type of lesion elicits.

Full-thickness articular cartilage defects include damage to thearticular cartilage, the underlying subchondral bone tissue, and thecalcified layer of cartilage located between the articular cartilage andthe subchondral bone. Full-thickness defects typically arise duringsevere trauma of the joint or during the late stages of degenerativejoint diseases, for example, during osteoarthritis. Since thesubchondral bone tissue is both innervated and vascularized, damage tothis tissue is often painful. The repair reaction induced by damage tothe subchondral bone usually results in the formation of fibrocartilageat the site of the full-thickness defect. Fibrocartilage, however, lacksthe biomechanical properties of articular cartilage and fails to persistin the joint on a long term basis.

Partial-thickness articular cartilage defects are restricted to thecartilage tissue itself. These defects usually include fissures orclefts in the articulating surface of the cartilage. Partial-thicknessdefects are caused by mechanical arrangements of the joint which in turninduce wearing of the cartilage tissue within the joint. In the absenceof innervation and vasculature, partial-thickness defects do not elicitrepair responses and therefore tend not to heal. Although painless,partial-thickness defects often degenerate into full-thickness defects.

Cartilage may develop abnormally or may be damaged by disease, such asrheumatoid arthritis or osteoarthritis, or by trauma, each of which canlead to physical deformity and debilitation. Whether cartilage isdamaged from trauma or congenital anomalies, its successful clinicalregeneration is often poor at best, as reviewed by Howell, et al.Osteoarthritis: Diagnosis and Management, 2nd ed., (Philadelphia, W.B.Saunders, 1990) and Kelley, et al. Textbook of Rheumatology, 3rd ed.,(Philadelphia, W.B. Saunders, 1989).

Bone conditions also are widespread. For example, there generally aretwo types of bone conditions: non-metabolic bone conditions, such asbone fractures, bone/spinal deformation, osteosarcoma, myeloma, bonedysplasia and scoliosis, and metabolic bone conditions, such asosteoporosis, osteomalacia, rickets, fibrous osteitis, renal bonedystrophy and Paget's disease of bone. Osteoporosis, a metabolic bonecondition, is a systemic disease characterized by increased bonefragility and fracturability due to decreased bone mass and change infine bone tissue structure, its major clinical symptoms including spinalkyphosis, and fractures of dorsolumbar bones, vertebral centra, femoralnecks, lower end of radius, ribs, upper end of humerus, and others. Inbone tissue, bone formation and destruction due to bone resorption occurconstantly. Upon deterioration of the balance between bone formation andbone destruction due to bone resorption, a quantitative reduction inbone occurs. Traditionally, bone resorption suppressors such asestrogens, calcitonin and bisphosphonates have been mainly used to treatosteoporosis.

Bone grafting is often used for the treatment of bone conditions.Indeed, more than 1.4 million bone grafting procedures are performed inthe developed world annually. Most of these procedures are administeredfollowing joint replacement surgery or during trauma surgicalreconstruction. The success or failure of bone grafting is dependentupon a number of factors including the vitality of the site of thegraft, the graft processing, and the immunological compatibility of theengrafted tissue.

In view of the prevalence of bone and cartilage conditions, novelsources of bone and cartilage tissue for therapeutic, diagnostic, andresearch uses are in high demand.

SUMMARY OF THE INVENTION

The invention is generally directed to postpartum-derived cells whichare derived from postpartum tissue which is substantially free of bloodand which is capable of self-renewal and expansion in culture and havingthe potential to differentiate into a cell of osteocyte or chondrocytephenotypes.

In some embodiments, the present invention provides cells derived fromhuman postpartum tissue substantially free of blood, capable ofself-renewal and expansion in culture, having the potential todifferentiate into a cell of an osteogenic or chondrogenic phenotype;requiring L-valine for growth; capable of growth in about 5% to about20% oxygen; and further having at least one of the followingcharacteristics:

production of at least one of GCP-2, tissue factor, vimentin, andalpha-smooth muscle actin;

lack of production of at least one of NOGO-A, GRO-alpha or oxidized lowdensity lipoprotein receptor, as detected by flow cytometry;

production of at least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C;

lack of production of at least one of CD31, CD34, CD45, CD80, CD86,CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flowcytometry;

expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is increasedfor at least one of interleukin 8; reticulon 1; chemokine (C—X—C motif)ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C—X—Cmotif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C—X—Cmotif) ligand 3; tumor necrosis factor, alpha-induced protein 3 orexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is increasedfor at least one of C-type lectin superfamily member A2, Wilms tumor 1,aldehyde dehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113;

expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is reducedfor at least one of: short stature homeobox 2; heat shock 27 kDa protein2; chemokine (C—X—C motif) ligand 12 (stromal cell-derived factor 1);elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa;

secretion of at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF,HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1;

lack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1b,I309, MDC, and VEGF, as detected by ELISA; and

the ability to undergo at least 40 population doublings in culture.

In certain embodiments, the postpartum-derived cell is an umbilicalcord-derived cell. In other embodiments, it is a placenta-derived cell.In specific embodiments, the cell has all identifying features of anyone of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074); celltype PLA 071003 (P11) (ATCC Accession No. PTA-6075); cell type PLA071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7)(ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCCAccession No. PTA-6068). The postpartum-derived cells of the inventionare preferably human cells.

The cells may be induced to differentiate to an osteogenic orchondrogenic phenotype. Methods for inducing differentiation ofpostpartum-derived cells of the invention are contemplated. For example,the cells may be induced to differentiate to a cell having an osteogenicor chondrogenic phenotype. Methods of inducing differentiation of thecells of the invention preferably involve exposing the cells to one ormore differentiation-inducing agents. Osteogenesis inducing agents ofthe invention include bone morphogenic proteins (e.g., BMP-2, BMP-4) andtransforming growth factor (TGF)-beta1, and combinations thereof.Chondrogenesis inducing agents of the invention include TGF-beta3,GDF-5, and a combination thereof. The invention includes thedifferentiation-induced cells and populations, compositions, andproducts thereof. Differentiation-induced cells of an osteogenic lineagepreferably express at least one osteogenic lineage marker (e.g.,osteocalcin, bone sialoprotein, alkaline phosphatase). Differentiationof PPDCs to an osteogenic lineage may be assessed by any means known inthe art, for example but not limited to, measurement of mineralization(e.g., von Kossa staining). Differentiation-induced cells of achondrogenic lineage preferably express at least one chondrogeniclineage marker (e.g., glycosaminoglycan, type II collagen).Differentiation of PPDCs to a chondrogenic lineage may be assessed byany means known in the art, for example but not limited to, Safranin-Oor hematoxylin/eosin staining.

Populations of PPDCs are provided by the invention. The PPDCs may bedifferentiation-induced or undifferentiated. In some embodiments, apopulation of postpartum-derived cells is mixed with another populationof cells. In some embodiments, the cell population is heterogeneous. Aheterogeneous cell population of the invention may comprise at leastabout 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%undifferentiated or differentiation-induced PPDCs of the invention. Theheterogeneous cell populations of the invention may further comprisebone marrow cells, stem cells, chondroblasts, chondrocytes, osteoblasts,osteocytes, osteoclasts, bone lining cells, or other bone or cartilagecell progenitors. Cell populations of the invention may be substantiallyhomogeneous, i.e., comprises substantially only PPDCs (preferably atleast about 96%, 97%, 98%, 99% or more PPDCs). The homogeneous cellpopulation of the invention may comprise umbilical cord- orplacenta-derived cells. Homogeneous populations of placenta-derivedcells may be of neonatal or maternal lineage. Homogeneity of a cellpopulation may be achieved by any method known in the art, for example,by cell sorting (e.g., flow cytometry), bead separation, or by clonalexpansion.

The invention also provides heterogeneous and homogeneous cell culturescontaining undifferentiated or differentiation-inducedpostpartum-derived cells of the invention.

Some embodiments of the invention provide a matrix for implantation intoa patient seeded with one or more postpartum-derived cells of theinvention or containing or treated with a cell lysate, conditionedmedium, or extracellular matrix thereof. The PPDCs may bedifferentiation-induced or undifferentiated. The matrix may contain orbe treated with one or more bioactive factors including anti-apoptoticagents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspaseinhibitors); anti-inflammatory agents (e.g., p38 MAPK inhibitors,TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine.

Also encompassed within the scope of the invention are PPDC-products,including extracellular matrices of PPDCs, cell lysates (e.g., solublecell fractions) of PPDCs, and PPDC-conditioned medium.

In some embodiments the invention provides compositions of PPDCs and oneor more bioactive factors, for example, but not limited to growthfactors, chondrogenic or osteogenic differentiation inducing factors,anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II,HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPKinhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors,PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine).

Pharmaceutical compositions of the postpartum-derived cells,extracellular matrix produced thereby, cell lysates thereof, andPPDC-conditioned medium are included within the scope of the invention.The pharmaceutical compositions preferably include a pharmaceuticallyacceptable carrier or excipient. The pharmaceutical compositions arepreferably for treating bone or cartilage conditions as defined herein.

The invention further provides in some aspects methods of regeneratingbone or cartilage tissue in a patient in need thereof by administeringcells, matrices, or PPDC-products of the invention into a patient areprovided.

Further provided by the invention are methods for treating a conditionsuch as a bone or cartilage condition in a patient by administering oneor more postpartum-derived cells, PPDC populations, matrices, celllysates, a combination of cell lysate and extracellular matrix,conditioned medium, or compositions of the invention. The PPDCs, whetherdifferentiated or undifferentiated, or a combination thereof,extracellular matrix produced thereby, cell lysates thereof, acombination of cell lysate and extracellular matrix, matrices (e.g.,scaffolds), conditioned medium, and compositions of the invention may beused in the treatment of bone or cartilage tissue conditions, forexample, but not limited to congenital defects, bone fractures, meniscalinjuries or defects, bone/spinal deformation, osteosarcoma, myeloma,bone dysplasia and scoliosis, osteoporosis, periodontal disease, dentalbone loss, osteomalacia, rickets, fibrous osteitis, renal bonedystrophy, spinal fusion, spinal disc reconstruction or removal, Paget'sdisease of bone, meniscal injuries, rheumatoid arthritis,osteoarthritis, or a traumatic or surgical injury to cartilage or bone.

Also provided by the invention are kits comprising thepostpartum-derived cells, PPDC-conditioned medium, lysate, and/orextracellular matrices of the invention. The kits of the invention mayfurther contain at least one component of a matrix, a second cell type,a hydrating agent, a ell culture substrate, a differentiation-inducingagent, cell culture media, and instructions, for example, for culture ofthe cells or administration of the cells and/or cell products.

In some embodiments, the invention provides methods for identifyingcompounds that modulate chondrogenesis or osteogenesis of apostpartum-derived cell comprising contacting a cell of the inventionwith said compound and monitoring the cell for a marker ofchondrogenesis or osteogenesis. Also provided are methods foridentifying compound toxic to a postpartum-derived cell of the inventionby contacting said cell with said compound and monitoring survival ofsaid cell.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent—able to give rise to all embryonic and extraembryonic celltypes; (2) pluripotent—able to give rise to all embryonic cell types;(3) multipotent—able to give rise to a subset of cell lineages, but allwithin a particular tissue, organ, or physiological system (for example,hematopoietic stem cells (HSC) can produce progeny that include HSC(self-renewal), blood cell-restricted oligopotent progenitors, and allcell types and elements (e.g., platelets) that are normal components ofthe blood); (4) oligopotent—able to give rise to a more restrictedsubset of cell lineages than multipotent stem cells; and (5)unipotent—able to give rise to a single cell lineage (e.g.,spermatogenic stem cells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself and, under normalcircumstances, differentiate to yield the specialized cell types of thetissue from which it originated, and possibly other tissue types. Anembryonic stem cell is a pluripotent cell from the inner cell mass of ablastocyst-stage embryo. A fetal stem cell is one that originates fromfetal tissues or membranes. A postpartum stem cell is a multipotent orpluripotent cell that originates substantially from extraembryonictissue available after birth, namely, the placenta and the umbilicalcord. These cells have been found to possess features characteristic ofpluripotent stem cells, including rapid proliferation and the potentialfor differentiation into many cell lineages. Postpartum stem cells maybe blood-derived (e.g., as are those obtained from umbilical cord blood)or non-blood-derived (e.g., as obtained from the non-blood tissues ofthe umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term committed,when applied to the process of differentiation, refers to a cell thathas proceeded in the differentiation pathway to a point where, undernormal circumstances, it will continue to differentiate into a specificcell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a mesodermal, ectodermalor endodermal lineage refers to a cell that becomes committed to aspecific mesodermal, ectodermal or endodermal lineage, respectively.Examples of cells that differentiate into a mesodermal lineage or giverise to specific mesodermal cells include, but are not limited to, cellsthat are adipogenic, chondrogenic, cardiogenic, dermatogenic,hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic,osteogenic, pericardiogenic, or stromal. Examples of cells thatdifferentiate into ectodermal lineage include, but are not limited toepidermal cells, neurogenic cells, and neurogliagenic cells. Examples ofcells that differentiate into endodermal lineage include, but are notlimited to pleurigenic cells, and hepatogenic cells, cell that give riseto the lining of the intestine, and cells that give rise to pancreogenicand splanchogenic cells.

The cells of the invention are referred to herein as postpartum-derivedcells (PPDCs). Subsets of the cells of the present invention arereferred to as placenta-derived cells (PDCs) or umbilical cord-derivedcells (UDCs). PPDCs of the invention encompass undifferentiated anddifferentiation-induced cells. In addition, the cells may be describedas being stem or progenitor cells, the latter term being used in thebroad sense. The term derived is used to indicate that the cells havebeen obtained from their biological source and grown or otherwisemanipulated in vitro (e.g., cultured in a growth medium to expand thepopulation and/or to produce a cell line). The in vitro manipulations ofpostpartum-derived cells and the unique features of thepostpartum-derived cells of the present invention are described indetail below.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture”). A primary cell culture is a cultureof cells, tissues or organs taken directly from organisms and before thefirst subculture. Cells are expanded in culture when they are placed ina growth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is sometimesmeasured by the amount of time needed for the cells to double in number.This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium, andtime between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. While the cells arecultured in the medium, they secrete cellular factors that can providetrophic support to other cells. Such trophic factors include, but arenot limited to hormones, cytokines, extracellular matrix (ECM),proteins, vesicles, antibodies, and granules. The medium containing thecellular factors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, proliferation, maturation, differentiation, and/ormaintenance of a cell, or stimulates increased activity of a cell.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

As used herein, the term Growth medium refers to a culture mediumsufficient for expansion of postpartum-derived cells. The culture mediumof Growth medium preferably contains Dulbecco's Modified Essential Media(DMEM). More preferably, Growth medium contains glucose. Growth mediumpreferably contains DMEM-low glucose (DMEM-LG) (Invitrogen, Carlsbad,Calif.). Growth medium preferably contains about 15% (v/v) serum (e.g.,fetal bovine serum, defined bovine serum). Growth medium preferablycontains at least one antibiotic agent and/or antimycotic agent (e.g.,penicillin, streptomycin, amphotericin B, gentamicin, nystatin;preferably, 50 units/milliliter penicillin G sodium and 50micrograms/milliliter streptomycin sulfate). Growth medium preferablycontains 2-mercaptoethanol (Sigma, St. Louis Mo.). Most preferably,Growth medium contains DMEM-low glucose, serum, 2-mercaptoethanol, andan antibiotic agent.

As used herein, standard growth conditions refers to standardatmospheric conditions comprising about 5% CO₂, a temperature of about35-39° C., more preferably 37° C., and a relative humidity of about100%.

The term isolated refers to a cell, cellular component, or a moleculethat has been removed from its native environment.

The term about refers to an approximation of a stated value within arange of ±10%.

Bone condition (or injury or disease) is an inclusive term encompassingacute and chronic and metabolic and non-metabolic conditions, disordersor diseases of bone. The term encompasses conditions caused by diseaseor trauma or failure of the tissue to develop normally. Examples of boneconditions include but are not limited to congenital bone defects, bonefractures, meniscal injuries or defects, bone/spinal deformation,osteosarcoma, myeloma, bone dysplasia and scoliosis, osteoporosis,periodontal disease, dental bone loss; osteomalacia, rickets, fibrousosteitis, renal bone dystrophy, spinal fusion, spinal discreconstruction or removal, and Paget's disease of bone.

Cartilage condition (or injury or disease) is an inclusive termencompassing acute and chronic conditions, disorders, or diseases ofcartilage. The term encompasses conditions including but not limited tocongenital defects, meniscal injuries, rheumatoid arthritis,osteoarthritis, or a traumatic or surgical injury to cartilage.

The term treating (or treatment of) a bone or cartilage condition refersto ameliorating the effects of, or delaying, halting or reversing theprogress of, or delaying or preventing the onset of, a bone or cartilagecondition as defined herein.

The term effective amount refers to a concentration of a reagent orpharmaceutical composition, such as a growth factor, differentiationagent, trophic factor, cell population or other agent, that is effectivefor producing an intended result, including cell growth and/ordifferentiation in vitro or in vivo, or treatment of a bone or cartilagecondition as described herein. With respect to growth factors, aneffective amount may range from about 1 nanogram/milliliter to about 1microgram/milliliter. With respect to PPDCs as administered to a patientin vivo, an effective amount may range from as few as several hundred orfewer to as many as several million or more. In specific embodiments, aneffective amount may range from 10³-10¹¹. It will be appreciated thatthe number of cells to be administered will vary depending on thespecifics of the disorder to be treated, including but not limited tosize or total volume/surface area to be treated, as well as proximity ofthe site of administration to the location of the region to be treated,among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term matrix as used herein refers to a support for the PPDCs of theinvention, for example, a scaffold (e.g., VICRYL, PCL/PGA, or RAD16) orsupporting medium (e.g., hydrogel, extracellular membrane protein (e.g.,MATRIGEL (BD Discovery Labware, Bedford, Mass.)).

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other complication commensurate with a reasonable benefit/risk ratio.As described in greater detail herein, pharmaceutically acceptablecarriers suitable for use in the present invention include liquids,semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds). Asused herein, the term biodegradable describes the ability of a materialto be broken down (e.g., degraded, eroded, dissolved) in vivo. The termincludes degradation in vivo with or without elimination (e.g., byresorption) from the body. The semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orby breakdown and elimination through natural pathways.

Several terms are used herein with respect to cell replacement therapy.The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy.

The following abbreviations are used herein:

ANG2 (or Ang2) for angiopoietin 2;

APC for antigen-presenting cells;

BDNF for brain-derived neurotrophic factor;

bFGF for basic fibroblast growth factor;

bid (BID) for “bis in die” (twice per day);

BSP for bone sialoprotein;

CK18 for cytokeratin 18;

CXC ligand 3 for chemokine receptor ligand 3;

DAPI for 4′-6-Diamidino-2-phenylindole-2HCl;

DMEM for Dulbecco's Minimal Essential Medium;

DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose;

EDTA for ethylene diamine tetraacetic acid;

EGF (or E) for epidermal growth factor;

EPO for erythropoietin;

FACS for fluorescent activated cell sorting;

FBS for fetal bovine serum;

FGF (or F) for fibroblast growth factor;

GCP-2 for granulocyte chemotactic protein-2;

GDF-5 for growth and differentiation factor 5;

GFAP for glial fibrillary acidic protein;

HB-EGF for heparin-binding epidermal growth factor;

HCAEC for Human coronary artery endothelial cells;

HGF for hepatocyte growth factor;

hMSC for Human mesenchymal stem cells;

HNF-1alpha for hepatocyte-specific transcription factor;

HUVEC for Human umbilical vein endothelial cells;

I309 for a chemokine and the ligand for the CCR8 receptor and isresponsible for chemoattraction of TH2 type T-cells;

IGF for insulin-like growth factor;

IL-6 for interleukin-6;

IL-8 for interleukin 8;

K19 for keratin 19;

K8 for keratin 8;

KGF for keratinocyte growth factor;

MCP-1 for monocyte chemotactic protein 1;

MDC for macrophage-derived chemokine;

MIP1alpha for macrophage inflammatory protein 1alpha;

MIP1beta for macrophage inflammatory protein 1beta;

MMP for matrix metalloprotease (MMP);

MSC for mesenchymal stem cells;

NHDF for Normal Human Dermal Fibroblasts;

NPE for Neural Progenitor Expansion media;

OxLDLR for oxidized low density lipoprotein receptor;

PBMC for peripheral blood mononuclear cell;

PBS for phosphate buffered saline;

PDC for placenta-derived cell;

PDGFbb for platelet derived growth factor;

PDGFr-alpha for platelet derived growth factor receptor alpha;

PD-L2 for programmed-death ligand 2;

PE for phycoerythrin;

PO for “per os” (by mouth);

PPDC for postpartum-derived cell;

Rantes (or RANTES) for regulated on activation, normal T cell expressedand secreted;

rb for rabbit;

rh for recombinant human;

SC for subcutaneously;

SCID for severe combined immunodeficiency;

SDF-1alpha for stromal-derived factor 1alpha;

SHH for sonic hedgehog;

SMA for smooth muscle actin;

SOP for standard operating procedure;

TARC for thymus and activation-regulated chemokine;

TCP for tissue culture plastic;

TGFbeta2 for transforming growth factor beta2;

TGFbeta-3 for transforming growth factor beta-3;

TIMP1 for tissue inhibitor of matrix metalloproteinase 1;

TPO for thrombopoietin;

TuJ1 for BIII Tubulin;

UDC for umbilical cord-derived cell;

VEGF for vascular endothelial growth factor;

vWF for von Willebrand factor; and

alphaFP for alpha-fetoprotein.

Description

Various patents and other publications are cited herein and throughoutthe specification, each of which is incorporated by reference herein inits entirety.

In one aspect, the invention provides postpartum-derived cells (PPDCs)derived from postpartum tissue substantially free of blood. The PPDCsmay be derived from placenta of a mammal including but not limited tohuman. The cells are capable of self-renewal and expansion in culture.The postpartum-derived cells have the potential to differentiate intocells of other phenotypes. The invention provides, in one of its severalaspects cells that are derived from umbilical cord, as opposed toumbilical cord blood. The invention also provides, in one of its severalaspects, cells that are derived from placental tissue.

The cells have been characterized as to several of their cellular,genetic, immunological, and biochemical properties. For example, thecells have been characterized by their growth by their cell surfacemarkers, by their gene expression, by their ability to produce certainbiochemical trophic factors, and by their immunological properties.

Derivation and Expansion of Postpartum-Derived Cells (PPDCs)

According to the methods described herein, a mammalian placenta andumbilical cord are recovered upon or shortly after termination of eithera full-term or pre-term pregnancy, for example, after expulsion afterbirth. Postpartum tissue can be obtained from any completed pregnancy,full-term or less than full-term, whether delivered vaginally, orthrough other means, for example, Cessarian section. The postpartumtissue may be transported from the birth site to a laboratory in asterile container such as a flask, beaker, culture dish, or bag. Thecontainer may have a solution or medium, including but not limited to asalt solution, such as, for example, Dulbecco's Modified Eagle's Medium(DMEM) or phosphate buffered saline (PBS), or any solution used fortransportation of organs used for transplantation, such as University ofWisconsin solution or perfluorochemical solution. One or more antibioticand/or antimycotic agents, such as but not limited to penicillin,streptomycin, amphotericin B, gentamicin, and nystatin, may be added tothe medium or buffer. The postpartum tissue may be rinsed with ananticoagulant solution such as heparin-containing solution. It ispreferable to keep the tissue at about 4-10° C. prior to extraction ofPPDCs. It is even more preferable that the tissue not be frozen prior toextraction of PPDCs.

Isolation of PPDCs preferably occurs in an aseptic environment. Bloodand debris are preferably removed from the postpartum tissue prior toisolation of PPDCs. For example, the postpartum tissue may be washedwith buffer solution, such as but not limited to phosphate bufferedsaline. The wash buffer also may comprise one or more antimycotic and/orantibiotic agents, such as but not limited to penicillin, streptomycin,amphotericin B, gentamicin, and nystatin.

In some aspects of the invention, the different cell types present inpostpartum tissue are fractionated into subpopulations from which thePPDCs can be isolated. This may be accomplished using techniques forcell separation including, but not limited to, enzymatic treatment todissociate postpartum tissue into its component cells, followed bycloning and selection of specific cell types, for example but notlimited to selection based on morphological and/or biochemical markers;selective growth of desired cells (positive selection), selectivedestruction of unwanted cells (negative selection); separation basedupon differential cell agglutinability in the mixed population as, forexample, with soybean agglutinin; freeze-thaw procedures; differentialadherence properties of the cells in the mixed population; filtration;conventional and zonal centrifugation; centrifugal elutriation(counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and flow cytometry, forexample, fluorescence activated cell sorting (FACS).

In a preferred embodiment, postpartum tissue comprising a whole placentaor a fragment or section thereof is disaggregated by mechanical force(mincing or shear forces), enzymatic digestion with single orcombinatorial proteolytic enzymes, such as a matrix metalloproteaseand/or neutral protease, for example, collagenase, trypsin, dispase,LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.), hyaluronidase,and/or pepsin, or a combination of mechanical and enzymatic methods. Forexample, the cellular component of the postpartum tissue may bedisaggregated by methods using collagenase-mediated dissociation.Enzymatic digestion methods preferably employ a combination of enzymes,such as a combination of a matrix metalloprotease and a neutralprotease. The matrix metalloprotease is preferably a collagenase. Theneutral protease is preferably thermolysin or dispase, and mostpreferably is dispase. More preferably, enzymatic digestion ofpostpartum tissue uses a combination of a matrix metalloprotease, aneutral protease, and a mucolytic enzyme for digestion of hyaluronicacid, such as a combination of collagenase, dispase, and hyaluronidaseor a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis,Ind.) and hyaluronidase. Collagenase may be type 1, 2, 3, or 4. Otherenzymes known in the art for cell isolation include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase, that may be used either on their own or in combination withother enzymes such as matrix metalloproteases, mucolytic enzymes, andneutral proteases. Serine proteases are preferably used consecutivelyfollowing use of other enzymes. The temperature and period of timetissues or cells are in contact with serine proteases is particularlyimportant. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNAse are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The degree of dilution of thedigestion may also greatly affect the cell yield as cells may be trappedwithin the viscous digest. The LIBERASE (Boehringer Mannheim Corp.,Indianapolis, Ind.) Blendzyme (Roche) series of enzyme combinations arevery useful and may be used in the instant methods. Other sources ofenzymes are known, and the skilled artisan may also obtain such enzymesdirectly from their natural sources. The skilled artisan is alsowell-equipped to assess new, or additional enzymes or enzymecombinations for their utility in isolating the cells of the invention.Preferred enzyme treatments are 0.5, 1, 1.5, or 2 hours long or longer.In more preferred embodiments, the tissue is incubated at 37° C. duringthe enzyme treatment of the disintegration step.

Postpartum tissue comprising the umbilical cord and placenta may be usedwithout separation. Alternatively, the umbilical cord may be separatedfrom the placenta by any means known in the art. In some embodiments ofthe invention, postpartum tissue is separated into two or more sections,such as umbilical cord and placenta. In some embodiments of theinvention, placental tissue is separated into two or more sections, eachsection consisting predominantly of either neonatal, neonatal andmaternal, or maternal aspect. The separated sections then aredissociated by mechanical and/or enzymatic dissociation according to themethods described herein. Cells of neonatal or maternal lineage may beidentified by any means known in the art, for example, by karyotypeanalysis or in situ hybridization for the Y-chromosome. Karyotypeanalysis also may be used to identify cells of normal karyotype.

Isolated cells or postpartum tissue from which PPDCs grow out may beused to initiate, or seed, cell cultures. Cells are transferred tosterile tissue culture vessels either uncoated or coated withextracellular matrix or ligands such as laminin, collagen, gelatin,fibronectin, ornithine, vitronectin, and extracellular membrane protein(e.g., MATRIGEL (BD Discovery Labware, Bedford, Mass.)). PPDCs arecultured in any culture medium capable of sustaining growth of the cellssuch as, but not limited to, DMEM (high or low glucose), Eagle's basalmedium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove'smodified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),DMEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), andCELL-GRO FREE. The culture medium may be supplemented with one or morecomponents including, for example, serum (e.g., fetal bovine serum(FBS), preferably about 2-15% (v/v); equine serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination. The culture mediumpreferably comprises Growth medium (DMEM-low glucose, serum, BME, anantimycotic agent, and an antibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. In a preferred embodiment, the cells are cultured at about 0 toabout 5 percent by volume CO₂ in air. In some preferred embodiments, thecells are cultured at about 2 to about 25 percent O₂ in air, preferablyabout 5 to about 20 percent O₂ in air. The cells preferably are culturedat about 25 to about 40° C., more preferably about 35° C. to about 39°C., and more preferably are cultured at 37° C. The cells are preferablycultured in an incubator. The medium in the culture vessel can be staticor agitated, for example, using a bioreactor. PPDCs preferably are grownunder low oxidative stress (e.g., with addition of glutathione, ascorbicacid, catalase, tocopherol, N-acetylcysteine). “Low oxidative stress”,as used herein, refers to conditions of no or minimal free radicaldamage to the cultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

The culture medium is changed as necessary, for example, by carefullyaspirating the medium from the dish, for example, with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulate in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to bePPDCs.

After culturing the cells or tissue fragments for a sufficient period oftime, PPDCs will have grown out, either as a result of migration fromthe postpartum tissue or cell division, or both. In some embodiments ofthe invention, PPDCs are passaged, or removed to a separate culturevessel containing fresh medium of the same or a different type as thatused initially, where the population of cells can be mitoticallyexpanded. PPDCs are preferably passaged up to about 100% confluence,more preferably about 70 to about 85% confluence. The lower limit ofconfluence for passage is understood by one skilled in the art. Theplacenta-derived cells of the invention may be utilized from the firstsubculture (passage 0) to senescence. The preferable number of passagesis that which yields a cell number sufficient for a given application.In certain embodiments, the cells are passaged 2 to 25 times, preferably4 to 20 times, more preferably 8 to 15 times, more preferably 10 or 11times, and most preferably 11 times. Cloning and/or subcloning may beperformed to confirm that a clonal population of cells has beenisolated.

Cells of the invention may be cryopreserved and/or stored prior to use.

Characterization of PPDCs

PPDCs may be characterized, for example, by growth characteristics(e.g., population doubling capability, doubling time, passages tosenescence), karyotype analysis (e.g., normal karyotype; maternal orneonatal lineage), flow cytometry (e.g., FACS analysis),immunohistochemistry and/or immunocytochemistry (e.g., for detection ofepitopes including but not limited to vimentin, desmin, alpha-smoothmuscle actin, cytokeratin 18, von Willebrand factor, CD34, GROalpha,GCP-2, oxidized low density lipoprotein receptor 1, and NOGO-A), geneexpression profiling (e.g., gene chip arrays; polymerase chain reaction(for example, reverse transcriptase PCR, real time PCR, and conventionalPCR)), protein arrays, protein secretion (e.g., by plasma clotting assayor analysis of PPDC-conditioned medium, for example, by Enzyme LinkedImmunoSorbent Assay (ELISA)), antibody analysis (e.g., ELISA; antibodystaining for cell surface markers including but not limited to CD10,CD13, CD31, CD34, CD44, CD45, CD73, CD80, CD86, CD90, CD117, CD141,CD178, platelet-derived growth factor receptor alpha (PDGFr-alpha), HLAclass I antigens (HLA-A, HLA-B, HLA-C), HLA class II antigens (HLA-DP,HLA-DQ, HLA-DR), B7-H2, and PD-L2), mixed lymphocyte reaction (e.g., asmeasure of stimulation of allogeneic PBMCs), and/or other methods knownin the art.

PPDCs can undergo at least 40 population doublings in culture.Population doubling may be calculated as [ln(cell final/cell initial)/ln2]. Doubling time may be calculated as (time in culture (h)/populationdoubling).

Undifferentiated PPDCs preferably produce of at least one of NOGO-A,GCP-2, tissue factor, vimentin, and alpha-smooth muscle actin; morepreferred are cells which produce each of GCP-2, tissue factor,vimentin, and alpha-smooth muscle actin. In some embodiments, two,three, four, or five of these factors are produced by the PPDCs.

In some embodiments, PPDCs lack production of at least one of NOGO-A,GRO-alpha, or oxidized low density lipoprotein receptor, as detected byflow cytometry. In some embodiments, PPDCs lack production of at leasttwo or three of these factors.

PPDCs may comprise at least one cell surface marker of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C. PPDCs preferably produceeach of these surface markers. PPDCs may be characterized in their lackof production of at least one of CD31, CD34, CD45, CD80, CD86, CD117,CD141, CD178, B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flowcytometry. PPDCs preferably lack production of each of these surfacemarkers.

In some embodiments, PPDCs exhibit expression, which relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, is increased for at least one of interleukin 8;reticulon 1; chemokine (C—X—C motif) ligand 1 (melanoma growthstimulating activity, alpha); chemokine (C—X—C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C—X—C motif) ligand 3;and tumor necrosis factor, alpha-induced protein 3; or at least one ofC-type lectin superfamily member A2, Wilms tumor 1, aldehydedehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113. Preferred PPDCs express, relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, increased levels of interleukin 8; reticulon 1;chemokine (C—X—C motif) ligand 1 (melanoma growth stimulating activity,alpha); chemokine (C—X—C motif) ligand 6 (granulocyte chemotacticprotein 2); chemokine (C—X—C motif) ligand 3; and tumor necrosis factor,alpha-induced protein 3; or increased levels of C-type lectinsuperfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 familymember A2, renin, oxidized low density lipoprotein receptor 1, proteinkinase C zeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013,downregulated in ovarian cancer 1, and clone DKFZp547K1113. In PPDCswherein expression, relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is increasedfor at least one of interleukin 8; reticulon 1; chemokine (C—X—C motif)ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C—X—Cmotif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C—X—Cmotif) ligand 3; and tumor necrosis factor, alpha-induced protein 3,increased relative levels of at least one of C-type lectin superfamilymember A2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2,renin, oxidized low density lipoprotein receptor 1, protein kinase Czeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013,downregulated in ovarian cancer 1, and clone DKFZp547K1113 arepreferably not present. In PPDCs wherein expression, relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, is increased for at least one of C-type lectinsuperfamily member A2, Wilms tumor 1, aldehyde dehydrogenase 1 familymember A2, renin, oxidized low density lipoprotein receptor 1, proteinkinase C zeta, clone IMAGE:4179671, hypothetical protein DKFZp564F013,downregulated in ovarian cancer 1, and clone DKFZp547K1113, increasedrelative levels of at least one of interleukin 8; reticulon 1; chemokine(C—X—C motif) ligand 1 (melanoma growth stimulating activity, alpha);chemokine (C—X—C motif) ligand 6 (granulocyte chemotactic protein 2);chemokine (C—X—C motif) ligand 3; and tumor necrosis factor,alpha-induced protein 3 are preferably not present.

PPDCs may have expression, which relative to a human cell that is afibroblast, a mesenchymal stem cell, or an ileac crest bone marrow cell,is reduced for at least one of: short stature homeobox 2; heat shock 27kDa protein 2; chemokine (C—X—C motif) ligand 12 (stromal cell-derivedfactor 1); elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022);mesenchyme homeobox 2; sine oculis homeobox homolog 1; crystallin, alphaB; dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa; the skilled artisan will appreciatethat the expression of a wide variety of genes is convenientlycharacterized on a gene array, for example on a Affymetrix GENECHIP.

PPDCs may secrete a variety of biochemically active factors, such asgrowth factors, chemokines, cytokines and the like. Preferred cellssecrete at least one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF,BDNF, TPO, MIP1a, RANTES, and TIMP1. PPDCs may be characterized in theirlack of secretion of at least one of TGF-beta2, ANG2, PDGFbb, MIP1b,I309, MDC, and VEGF, as detected by ELISA. These and othercharacteristics are available to identify and characterize the cells,and distinguish the cells of the invention from others known in the art.

In preferred embodiments, the cell comprises two or more of theforegoing characteristics. More preferred are those cells comprising,three, four, or five or more of the characteristics. Still morepreferred are those postpartum-derived cells comprising six, seven, oreight or more of the characteristics. Still more preferred presently arethose cells comprising all nine of the claimed characteristics.

Also presently preferred are cells that produce at least two of GCP-2,NOGO-A, tissue factor, vimentin, and alpha-smooth muscle actin. Morepreferred are those cells producing three, four, or five of theseproteins.

The skilled artisan will appreciate that cell markers are subject tovary somewhat under vastly different growth conditions, and thatgenerally herein described are characterizations in Growth Medium, orvariations thereof. Postpartum-derived cells that produce of at leastone, two, three, or four of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C are preferred. More preferred are those cellsproducing five, six, or seven of these cell surface markers. Still morepreferred are postpartum-derived cells that can produce eight, nine, orten of the foregoing cell surface marker proteins.

PPDCs that lack of production of at least one, two, three, or four ofthe proteins CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2,HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry are preferred.PPDCs lacking production of at least five, six, seven, or eight or moreof these markers are preferred. More preferred are cells which lackproduction of at least nine or ten of the cell surface markers. Mosthighly preferred are those cells lacking production of eleven, twelve,or thirteen of the foregoing identifying proteins.

Presently preferred cells produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

It is preferred that postpartum-derived cells exhibit expression, whichrelative to a human cell that is a fibroblast, a mesenchymal stem cell,or an ileac crest bone marrow cell, is increased for at least one of atleast one, two, or three of interleukin 8; reticulon 1; chemokine (C—X—Cmotif) ligand 1 (melanoma growth stimulating activity, alpha); chemokine(C—X—C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C—X—C motif) ligand 3; and tumor necrosis factor, alpha-induced protein3; or at least one, two, or three of C-type lectin superfamily memberA2, Wilms tumor 1, aldehyde dehydrogenase 1 family member A2, renin,oxidized low density lipoprotein receptor 1, protein kinase C zeta,clone IMAGE:4179671, hypothetical protein DKFZp564F013, downregulated inovarian cancer 1, and clone DKFZp547K1113. More preferred are thosecells which exhibit elevated relative expression of four or five, andstill more preferred are cell capable of increased relative expressionof six, seven, or eight of the foregoing genes of the respective genesets. Most preferably, the cells exhibit expression, which relative to ahuman cell that is a fibroblast, a mesenchymal stem cell, or an ileaccrest bone marrow cell, is increased for a combination of interleukin 8;reticulon 1; chemokine (C—X—C motif) ligand 1 (melanoma growthstimulating activity, alpha); chemokine (C—X—C motif) ligand 6(granulocyte chemotactic protein 2); chemokine (C—X—C motif) ligand 3;tumor necrosis factor, alpha-induced protein 3 or a combination ofC-type lectin superfamily member A2, Wilms tumor 1, aldehydedehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113.

For some embodiments, preferred are cells, which relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, have reduced expression for at least one of the genescorresponding to: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C—X—C motif) ligand 12 (stromal cell-derived factor 1);elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa. More preferred are cells that have,relative to human fibroblasts, mesenchymal stem cells, or ileac crestbone marrow cells, reduced expression of at least 5, 10, 15 or 20 genescorresponding to those listed above. Presently more preferred are cellwith reduced expression of at least 25, 30, or 35 of the genescorresponding to the listed sequences. Also more preferred are thosepostpartum-derived cells having expression that is reduced, relative tothat of a human fibroblast, a mesenchymal stem cell, or an ileac crestbone marrow cell, of genes corresponding to 35 or more, 40 or more, oreven all of the sequences listed.

Secretion of certain growth factors and other cellular proteins can makecells of the invention particularly useful. Preferred postpartum-derivedcells secrete at least one, two, three or four of MCP-1, IL-6, IL-8,GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, and TIMP1. Cellswhich secrete five, six, seven or eight of the listed proteins are alsopreferred. Cells which can secrete at least nine, ten, eleven or more ofthe factors are more preferred, as are cells which can secrete twelve ormore, or even all thirteen of the proteins in the foregoing list.

While secretion of such factors is useful, PPDCs can also becharacterized by their lack of secretion of factors into the medium.Postpartum-derived cells that lack secretion of at least one, two, threeor four of TGF-beta2, ANG2, PDGFbb, MIP1b, I309, MDC, and VEGF, asdetected by ELISA, are presently preferred for use. Cells that arecharacterized in their lack secretion of five or six of the foregoingproteins are more preferred. Cells which lack secretion of all seven ofthe factors listed above are also preferred.

Examples of placenta-derived cells of the invention were deposited withthe American Type Culture Collection (ATCC, Manassas, Va.) and assignedATCC Accession Numbers as follows: (1) strain designation PLA 071003(P8) was deposited Jun. 15, 2004 and assigned Accession No. PTA-6074;(2) strain designation PLA 071003 (P11) was deposited Jun. 15, 2004 andassigned Accession No. PTA-6075; and (3) strain designation PLA 071003(P16) was deposited Jun. 16, 2004 and assigned Accession No. PTA-6079.

Examples of umbilical cord-derived cells of the invention were depositedwith the American Type Culture Collection (ATCC, Manassas, Va.) on Jun.10, 2004, and assigned ATCC Accession Numbers as follows: (1) straindesignation UMB 022803 (P7) was assigned Accession No. PTA-6067; and (2)strain designation UMB 022803 (P17) was assigned Accession No. PTA-6068.

PPDCs can be isolated. The invention also provides compositions ofPPDCs, including populations of PPDCs. In some embodiments, the cellpopulation is heterogeneous. A heterogeneous cell population of theinvention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% PPDCs of the invention. In some embodiments, theheterogeneous cell populations of the invention may further comprisebone marrow cells, stem cells, chondroblasts, chondrocytes, and/orprogenitor cells. In some embodiments, the heterogeneous cellpopulations of the invention may further comprise bone marrow cells,stem cells, osteoblasts, osteocytes, osteoclasts, bone lining cells,and/or progenitor cells. In some embodiments, the population issubstantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). Thehomogeneous cell population of the invention may comprise umbilicalcord- or placenta-derived cells. Homogeneous populations of umbilicalcord-derived cells may be free of cells of maternal lineage. Homogeneouspopulations of placenta-derived cells may be of neonatal or maternallineage. Homogeneity of a cell population may be achieved by any methodknown in the art, for example, by cell sorting (e.g., flow cytometry),bead separation, or by clonal expansion.

Methods of the invention further include methods for producing apopulation of postpartum-derived cells by expanding a cell of theinvention in culture. The postpartum-derived cells of the inventionpreferably expand in the presence of from about 5% to about 20% oxygen.The postpartum-derived cells of the invention preferably are expanded inculture medium such as but not limited to Dulbecco's modified Eagle'smedium (DMEM), mesenchymal stem cell growth medium, advanced DMEM(Gibco), DMEM/MCDB201 (Sigma), RPMI1640, CELL-GRO FREE, advanced DMEM(Gibco), DMEM/MCDB201 (Sigma), Ham's F10 medium, Ham's F12 medium,DMEM/F12, Iscove's modified Dulbecco's medium, or Eagle's basal medium.The culture medium preferably contains low or high glucose, about 2%-15%(v/v) serum, betamercaptoethanol, and an antibiotic agent. The culturemedium may contain at least one of fibroblast growth factor,platelet-derived growth factor, vascular endothelial growth factor, andepidermal growth factor. The cells of the invention may be grown on anuncoated or coated surface. Surfaces for growth of the cells may becoated for example with gelatin, collagen (e.g., native or denatured),fibronectin, laminin, ornithine, vitronectin, or extracellular membraneprotein (e.g., MATRIGEL). In some embodiments, a population ofpostpartum-derived cells is mixed with another population of cells.

Culture of PPDCs in a Chondrogenic Medium

PPDCs may be induced to differentiate into a chondrogenic lineage bysubjecting them to differentiation-inducing cell culture conditions.PPDCs may be cultured in a chondrogenic medium comprising specificexogenous chondrogenic growth factors (e.g., in culture), such as, forexample, one or more of GDF-5 or transforming growth factor beta3(TGF-beta3), with or without ascorbate.

Preferred chondrogenic medium is supplemented with an antibiotic agent,amino acids including proline and glutamine, sodium pyruvate,dexamethasone, ascorbic acid, and insulin/tranferrin/selenium.Chondrogenic medium is preferably supplemented with sodium hydroxideand/or collagen. Most preferably, chondrogenic culture medium issupplemented with collagen. The cells may be cultured at high or lowdensity. Cells are preferably cultured in the absence of serum.

Culture of PPDCs in an Osteogenic Medium

PPDCs may be induced to differentiate into an osteogenic lineage bysubjecting them to differentiation-inducing cell culture conditions. Insome embodiments, PPDCs are cultured in osteogenic medium such as, butnot limited to, media (e.g., DMEM-low glucose) containing about 10⁻⁷molar and about 10⁻⁹ molar dexamethasone in combination with about 10micromolar to about 50 micromolar ascorbate phosphate salt (e.g.,ascorbate-2-phosphate) and between about 10 nanomolar and about 10millimolar beta-glycerophosphate. The medium preferably includes serum(e.g., bovine serum, horse serum). Osteogenic medium also may compriseone or more antibiotic/antimycotic agents. The osteogenic medium ispreferably supplemented with transforming growth factor-beta (e.g.,TGF-beta1) and/or bone morphogenic protein (e.g., BMP-2, BMP-4, or acombination thereof; most preferably BMP-4)

Assessment of Differentiation

PPDCs may be induced to differentiate to an ectodermal, endodermal, ormesodermal lineage. Methods to characterize differentiated cells thatdevelop from the PPDCs of the invention, include, but are not limitedto, histological, morphological, biochemical and immunohistochemicalmethods, or using cell surface markers, or genetically or molecularly,or by identifying factors secreted by the differentiated cell, and bythe inductive qualities of the differentiated PPDCs.

Chondrogenic differentiation may be assessed, for example, by Safranin-Ostaining for glycosaminoglycan expression by the cells orhematoxylin/eosin staining or by detection of a chondrogenic lienagemarker (e.g., sulfated glycosaminoglycans and proteoglycans, keratin,chondroitin, Type II collagen) in the culture or more preferably in thecells themselves.

PPDCs may be analyzed for an osteogenic phenotype by any method known inthe art, e.g., von Kossa staining or by detection of osteogenic markerssuch as osteocalcin, bone sialoprotein, alkaline phosphatase,osteonectin, osteopontin, type I collagen, bone morphogenic proteins,and/or core binding factor a1 in the culture or more preferably in thecells themselves.

Methods of Using PPDCs or Components or Products Thereof

Genetic Engineering of PPDCs

The cells of the invention can be engineered using any of a variety ofvectors including, but not limited to, integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors; non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Othermethods of introducing DNA into cells include the use of liposomes,electroporation, a particle gun, or by direct DNA injection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by or in operative association with, one or more appropriateexpression control elements such as promoter or enhancer sequences,transcription terminators, polyadenylation sites, among others, and aselectable marker.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines.

This method can be advantageously used to engineer cell lines whichexpress the gene product.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include, but are not limited to, the CMVpromoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus or elastingene promoter. Preferably, the control elements used to controlexpression of the gene of interest should allow for the regulatedexpression of the gene so that the product is synthesized only whenneeded in vivo. If transient expression is desired, constitutivepromoters are preferably used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary.

Inducible promoters include, but are not limited to, those associatedwith metallothionein and heat shock proteins.

The cells of the invention may be genetically engineered to “knock out”or “knock down” expression of factors that promote inflammation orrejection at the implant site. Negative modulatory techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation,” as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to a chondrocyte or osteocyte can be reduced or knocked outusing a number of techniques including, for example, inhibition ofexpression by inactivating the gene completely (commonly termed“knockout”) using the homologous recombination technique. Usually, anexon encoding an important region of the protein (or an exon 5′ to thatregion) is interrupted by a positive selectable marker, e.g., neo,preventing the production of normal mRNA from the target gene andresulting in inactivation of the gene. A gene may also be inactivated bycreating a deletion in part of a gene, or by deleting the entire gene.By using a construct with two regions of homology to the target genethat are far apart in the genome, the sequences intervening the tworegions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci.U.S.A. 88:3084).

Antisense, small interfering RNA, DNAzymes and ribozyme molecules whichinhibit expression of the target gene can also be used in accordancewith the invention to reduce the level of target gene activity. Forexample, antisense RNA molecules which inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Still further, triple helixmolecules can be utilized in reducing the level of target gene activity.

These techniques are described in detail by L. G. Davis et al. (eds),1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed., Appleton & Lange,Norwalk, Conn., which is incorporated herein by reference.

IL-1 is a potent stimulator of cartilage resorption and of theproduction of inflammatory mediators by chondrocytes (Campbell et al.,1991, J. Immun. 147: 1238). Using any of the foregoing techniques, theexpression of IL-1 can be knocked out or knocked down in the cells ofthe invention to reduce the risk of resorption of implanted cartilage orthe production of inflammatory mediators by the cells of the invention.Likewise, the expression of MHC class II molecules can be knocked out orknocked down in order to reduce the risk of rejection of the implantedtissue.

Once the cells of the invention have been genetically engineered, theymay be directly implanted into the patient to allow for the treatment ofa bone or cartilage condition, for example, rheumatoid or joint disease,or to produce an anti-inflammatory gene product such as, for example,peptides or polypeptides corresponding to the idiotype of neutralizingantibodies for GM-CSF, TNF, IL-1, IL-2, or other inflammatory cytokines.

Alternatively, the genetically engineered cells may be used to producenew tissue in vitro, which is then implanted in the subject, asdescribed herein.

Secretion of Trophic Factors

The secretion of growth factors by PPDCs may provide trophic support fora second cell type in vitro or in vivo. PPDCs may secrete, for example,at least one of monocyte chemotactic protein 1 (MCP-1), interleukin-6(IL6), interleukin 8 (IL-8), GCP-2, hepatocyte growth factor (HGF),keratinocyte growth factor (KGF), fibroblast growth factor (FGF),heparin binding epidermal growth factor (HB-EGF), brain-derivedneurotrophic factor (BDNF), thrombopoietin (TPO), macrophageinflammatory protein 1 alpha (MIP1a), RANTES, and tissue inhibitor ofmatrix metalloproteinase 1 (TIMP1), which can be augmented by a varietyof techniques, including ex vivo cultivation of the cells in chemicallydefined medium.

In some aspects of the invention, a population of PPDCs supports thesurvival, proliferation, growth, maintenance, maturation,differentiation, or increased activity of cells including stem cells,such as embryonic stem cells, bone marrow cells, chondrocytes,chondroblasts, and mixtures thereof. In some aspects of the invention, apopulation of PPDCs supports cells including stem cells, such asembryonic stem cells, bone marrow cells, osteoblasts, osteocytes,osteoclasts, bone lining cells, and mixtures thereof. In some aspects ofthe invention, a population of PPDCs supports cells including stemcells, such as embryonic stem cells, bone marrow cells, chondrocytes,chondroblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells,and mixtures thereof. In other embodiments, the population issubstantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs).

PPDCs have the ability to support survival, growth, and differentiationof other cell types in co-culture. In some embodiments, PPDCs areco-cultured in vitro to provide trophic support to other cells,including but not limited to stem cells, osteocytes, osteoblasts,osteoclasts, bone lining cells, chondrocytes, chondroblasts, and/or bonemarrow cells, or combinations thereof. For co-culture, it may bedesirable for the PPDCs and the desired other cells to be co-culturedunder conditions in which the two cell types are in contact. This can beachieved, for example, by seeding the cells as a heterogeneouspopulation of cells in culture medium or onto a suitable culturesubstrate. Alternatively, the PPDCs can first be grown to confluence andemployed as a substrate for the second desired cell type in culture. Inthis latter embodiment, the cells may further be physically separated,e.g., by a membrane or similar device, such that the other cell type maybe removed and used separately following the co-culture period. In otherembodiments, the desired other cells are cultured in contact with theconditioned medium, extracellular matrix, and/or cell lysate of thePPDCs. Use of PPDCs in co-culture to promote expansion anddifferentiation of other cell types may find applicability in researchand in clinical/therapeutic areas. For instance, PPDC co-culture may beutilized to facilitate growth and differentiation of cells of a givenphenotype in culture, for example, chondrocytes or osteocytes, for basicresearch purposes or for use in drug screening assays, for example. PPDCco-culture may also be utilized for ex vivo expansion of cells of anosteogenic or chondrogenic phenotype for later administration fortherapeutic purposes. For example, cells may be harvested from anindividual, expanded ex vivo in co-culture with PPDCs, then returned tothat individual (autologous transfer) or another individual (syngeneicor allogeneic transfer). In these embodiments, it will be appreciatedthat, following ex vivo expansion, the mixed population of cellscomprising the PPDCs could be administered to a patient in need oftreatment, for example, of a bone or cartilage condition as describedherein. Alternatively, in situations where autologous transfer isappropriate or desirable, the co-cultured cell populations may bephysically separated in culture, enabling removal of the autologouscells for administration to the patient.

Conditioned Medium of PPDCs

Another embodiment of the invention features use of PPDCs for productionof conditioned medium, either from undifferentiated PPDCs or from PPDCsincubated under conditions that stimulate differentiation into achondrogenic or osteogenic lineage. Such conditioned media arecontemplated for use in in vitro or ex vivo culture of cells, forexample, stem or progenitor cells, including but not limited to bonemarrow cells, osteoblasts, osteocytes, osteoclasts, bone lining cells,chondroblasts, and chondrocytes, or in vivo to support transplantedcells comprising homogeneous or heterogeneous populations of PPDCsand/or stem cells, osteocytes, osteoblasts, osteoclasts, bone liningcells, chondrocytes, chondroblasts, and bone marrow cells, for example.

Therapeutic Applications of PPDCs

PPDCs of the invention may be used to treat patients requiring therepair or replacement of cartilage or bone tissue resulting from diseaseor trauma or failure of the tissue to develop normally, or to provide acosmetic function, such as to augment facial or other features of thebody. Treatment may entail the use of the cells of the invention toproduce new cartilage tissue or bone tissue. For example, theundifferentiated or chondrogenic differentiation-induced cells of theinvention may be used to treat a cartilage condition, for example,rheumatoid arthritis or osteoarthritis or a traumatic or surgical injuryto cartilage. As another example, the undifferentiated or osteogenicdifferentiation-induced cells of the invention may be used to treat boneconditions, including metabolic and non-metabolic bone diseases.Examples of bone conditions include meniscal tears, spinal fusion,spinal disc removal, spinal reconstruction, bone fractures, bone/spinaldeformation, osteosarcoma, myeloma, bone dysplasia, scoliosis,osteoporosis, periodontal disease, dental bone loss, osteomalacia,rickets, fibrous osteitis, renal bone dystrophy, and Paget's disease ofbone.

The cells of the invention may be administered alone or as admixtureswith other cells. Cells that may be administered in conjunction withPPDCs include, but are not limited to, other multipotent or pluripotentcells or chondrocytes, chondroblasts, osteocytes, osteoblasts,osteoclasts, bone lining cells, stem cells, or bone marrow cells. Thecells of different types may be admixed with the PPDCs immediately orshortly prior to administration, or they may be co-cultured together fora period of time prior to administration.

The PPDCs may be administered with other beneficial drugs or biologicalmolecules (growth factors, trophic factors). When PPDCs are administeredwith other agents, they may be administered together in a singlepharmaceutical composition, or in separate pharmaceutical compositions,simultaneously or sequentially with the other agents (either before orafter administration of the other agents). Bioactive factors which maybe co-administered include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPOXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine) as well aslocal anesthetics. As another example, the cells may be co-administeredwith scar inhibitory factor as described in U.S. Pat. No. 5,827,735,incorporated herein by reference.

In one embodiment, PPDCs are administered as undifferentiated cells,i.e., as cultured in Growth Medium. Alternatively, PPDCs may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired phenotype, for example, a chondrogenicor osteogenic phenotype.

The cells of the invention may be surgically implanted, injected,delivered (e.g., by way of a catheter or syringe), or otherwiseadministered directly or indirectly to the site in need of repair oraugmentation. The cells may be administered by way of a matrix (e.g., athree-dimensional scaffold). The cells may be administered withconventional pharmaceutically acceptable carriers. Routes ofadministration of the cells of the invention or compositions orcomponents (e.g., ECM, cell lysate, conditioned medium) thereof includeintramuscular, ophthalmic, parenteral (including intravenous),intraarterial, subcutaneous, oral, and nasal administration. Particularroutes of parenteral administration include, but are not limited to,intramuscular, subcutaneous, intraperitoneal, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracisternal,intraspinal and/or peri-spinal routes of administration.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. Liquid or fluid pharmaceutical compositions,however, may be administered to a more general location (e.g.,throughout a diffusely affected area, for example), from which theymigrate to a particular location, e.g., by responding to chemicalsignals.

Other embodiments encompass methods of treatment by administeringpharmaceutical compositions comprising PPDC cellular components (e.g.,cell lysates or components thereof) or products (e.g., extracellularmatrix, trophic and other biological factors produced naturally by PPDCsor through genetic modification, conditioned medium from PPDC culture).Again, these methods may further comprise administering other activeagents, such as anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO,IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents(e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs(non-steroidal anti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN,SUPROFEN); immunosupressive/immunomodulatory agents (e.g., calcineurininhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g.,SIROLIMUS, EVEROLIMUS); anti-proliferatives (e.g., azathioprine,mycophenolate mofetil); corticosteroids (e.g., prednisolone,hydrocortisone); antibodies such as monoclonal anti-IL-2Ralpha receptorantibodies (e.g., basiliximab, daclizumab), polyclonal anti-T-cellantibodies (e.g., anti-thymocyte globulin (ATG); anti-lymphocyteglobulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), localanesthetics, and scar inhibitory factor as described in U.S. Pat. No.5,827,735, incorporated herein by reference.

Dosage forms and regimes for administering PPDCs or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the condition beingtreated, age, sex, body weight and general medical condition, and otherfactors known to medical practitioners. Thus, the effective amount of apharmaceutical composition to be administered to a patient is determinedby these considerations as known in the art.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of celltherapy with PPDCs. In addition, PPDCs have been shown not to stimulateallogeneic PBMCs in a mixed lymphocyte reaction. Accordingly,transplantation with allogeneic, or even xenogeneic, PPDCs may betolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. PPDCs may be encapsulated in a capsulethat is permeable to nutrients and oxygen required by the cell andtherapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, PPDCs may be genetically modifiedto reduce their immunogenicity.

Survival of transplanted PPDCs in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the target tissue, andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for cells of a specificlineage. Transplanted cells can also be identified by priorincorporation of tracer dyes such as rhodamine- or fluorescein-labeledmicrospheres, fast blue, bisbenzamide, ferric microparticles, orgenetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted PPDCs into a subject can beassessed by examining restoration of the function that was damaged ordiseased, for example, restoration of joint or bone function, oraugmentation of function.

Compositions and Pharmaceutical Compositions

Compositions of PPDCs and related products (e.g., extracellular matrix,lysate, soluble cell fraction, conditioned medium), including forexample pharmaceutical compositions, are included within the scope ofthe invention. Compositions of the invention may include one or morebioactive factors, for example but not limited to a growth factor, adifferentiation-inducing factor, a cell survival factor such as caspaseinhibitor, an anti-inflammatory agent such as p38 kinase inhibitor, oran angiogenic factor such as VEGF or bFGF. Some examples of bioactivefactors include PDGF-bb, EGF, bFGF, IGF-1, and LIF. In some embodiments,undifferentiated or differentiation-induced PDPCs are cultured incontact with the bioactive factor. In some embodiments, undifferentiatedPPDCs remain undifferentiated upon contact with the bioactive factor. Inother embodiments, the bioactive factor induces differentiation of thePPDCs.

Pharmaceutical compositions of the invention may comprise homogeneous orheterogeneous populations of PPDCs, extracellular matrix or cell lysatethereof, or conditioned medium thereof in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers for the cells of theinvention include organic or inorganic carrier substances suitable whichdo not deleteriously react with the cells of the invention orcompositions or components thereof. To the extent they arebiocompatible, suitable pharmaceutically acceptable carriers includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring. Pharmaceutical carriers suitable for use in the presentinvention are known in the art and are described, for example, inPharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton, Pa.) and WO96/05309, each of which are incorporated by reference herein.

The dosage (e.g., the number of cells to be administered) and frequencyof administration of the pharmaceutical compositions of the inventionwill depend upon a number of factors, including but not limited to, thenature of the condition to be treated, the extent of the symptoms of thecondition, characteristics of the patient (e.g., age, size, gender,health).

For example but not by way of limitation, PPDCs, extracellular matrix orcell lysates thereof, conditioned medium, compositions, and matricesproduced according to the invention can be used to repair or replacedamaged or destroyed cartilage tissue, to augment existing cartilagetissue, to introduce new or altered tissue, to modify artificialprostheses, or to join biological tissues or structures. For example,some embodiments of the invention would include (i) hip prosthesescoated with replacement cartilage tissue constructs grown inthree-dimensional cultures; (ii) knee reconstruction with cartilagetissue constructs; (iii) prostheses of other joints requiringreconstruction and/or replacement of articular cartilage; and (iv)cosmetic reconstruction with cartilage tissue constructs.

For example, the evaluation of internal derangements of articularcartilage in for example, the knee, hip, elbow, ankle and theglenohumeral joint, may be performed by arthroscopic techniques. In someembodiments, the injured or deteriorated portion of cartilage tissue isremoved, for example, by arthroscopic surgery, followed by cartilagegrafting. Cartilage tissue constructs may also be employed inreconstructive surgery for different types of joints. Detailedprocedures have been described in Resnick, D., and Niwayama, G., (eds),1988, Diagnosis of Bone and Joint Disorders, 2d ed., W.B. Sanders Co.,which is incorporated herein by reference.

Repair or replacement of damaged cartilage may be enhanced by fixationof the implanted cells and/or cartilage tissue at the site of repair.Various methods can be used to fix the new cells and/or cartilage tissuein place, including: patches derived from biocompatible tissues, whichcan be placed over the site; bioabsorbable sutures or other fasteners,e.g., pins, staples, tacks, screws and anchors; non-absorbable fixationdevices, e.g., sutures, pins, screws and anchors; adhesives.

As another example but not by way of limitation, PPDCs, extracellularmatrix or cell lysates thereof, conditioned medium, and the bone tissueproduced according to the invention can be used to repair or replacedamaged or destroyed bone tissue, to augment existing bone tissue, tointroduce new or altered tissue, or to modify artificial prostheses. Thecells of the invention may be administered alone, in a pharmaceuticallyacceptable carrier, or seeded on or in a matrix as described herein.

Use of PPDCs for Transplantation

The treatment methods of the subject invention involve the implantationof PPDCs or trans-differentiated cells into individuals in need thereof.The cells of the present invention may be allogeneic or autologous andmay be delivered to the site of therapeutic need or “home” to the site.

The cells of the present invention may be differentiated in vitro priorto implantation in a patient. In vitro differentiation allows forcontrolled application of bioactive factors.

The cells of the present invention may be induced to differentiate insitu or may be introduced in vivo to provide trophic support toendogenous cells. The appropriate cell implantation dosage in humans canbe determined from existing information relating to either the activityof the cells or the density of cells for bone or cartilage replacement.This information is also useful in calculating an appropriate dosage ofimplanted material. Additionally, the patient can be monitored todetermine if additional implantation can be made or implanted materialreduced accordingly.

To enhance the differentiation, survival or activity of implanted cells,additional bioactive factors may be added including growth factors,anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II,HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38 MAPKinhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors,PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPOXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine), and local anesthetics. To enhancevascularization and survival of transplanted bone tissue, angiogenicfactors such as VEGF, PDGF or bFGF can be added either alone or incombination with endothelial cells or their precursors including CD34+,CD34+/CD117+ cells.

Alternatively, PPDCs to be transplanted may be genetically engineered toexpress such growth factors, antioxidants, antiapoptotic agents,anti-inflammatory agents, or angiogenic factors.

PPDCs can be used to treat diseases or conditions of bone or cartilageor to augment or replace bone or cartilage. The disease or conditions tobe treated include but are not limited to osteoarthritis, osteoporosis,rheumatoid arthritis, chondrosis deformans, dental and oral cavitydisease (e.g., tooth fracture and defects), joint replacement,congenital abnormalities, bone fracture, and tumors (benign andmalignant).

One or more other components may be added to transplanted cells,including selected extracellular matrix components, such as one or moretypes of collagen known in the art, and/or growth factors, platelet-richplasma, and drugs. Alternatively, the cells of the invention may begenetically engineered to express and produce for growth factors.Details on genetic engineering of the cells of the invention areprovided infra. Bioactive factors which may be usefully incorporatedinto the cell formulation include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPOXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), and localanesthetics. For example, the cells may be co-administered with scarinhibitory factor as described in U.S. Pat. No. 5,827,735, incorporatedherein by reference.

Formulation of PPDCs for Transplantation

In a non-limiting embodiment, a formulation comprising the cells of theinvention is prepared for administration directly to the site where theproduction of new cartilage or bone tissue is desired. For example, andnot by way of limitation, the cells of the invention may be suspended ina hydrogel solution for injection. Examples of suitable hydrogels foruse in the invention include self-assembling peptides, such as RAD16.Alternatively, the hydrogel solution containing the cells may be allowedto harden, for instance in a mold, to form a matrix having cellsdispersed therein prior to implantation. Or, once the matrix hashardened, the cell formations may be cultured so that the cells aremitotically expanded prior to implantation. The hydrogel is an organicpolymer (natural or synthetic) which is cross-linked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure which entraps water molecules to form a gel. Examples ofmaterials which can be used to form a hydrogel include polysaccharidessuch as alginate and salts thereof, peptides, polyphosphazines, andpolyacrylates, which are crosslinked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively. In some embodiments, thesupport for the PPDCs of the invention is biodegradable.

In some embodiments of the invention, the formulation comprises an insitu polymerizable gel, as described, for example, in U.S. PatentApplication Publication 2002/0022676; Anseth et al., J. Control Release,78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22):3969-80(2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

Biodegradable polyphosphazenes have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl.

Hydrolysis of the side chain results in erosion of the polymer. Examplesof hydrolyzing side chains are unsubstituted and substituted imidizolesand amino acid esters in which the group is bonded to the phosphorousatom through an amino linkage (polyphosphazene polymers in which both Rgroups are attached in this manner are known as polyaminophosphazenes).For polyimidazolephosphazenes, some of the “R” groups on thepolyphosphazene backbone are imidazole rings, attached to phosphorous inthe backbone through a ring nitrogen atom. Other “R” groups can beorganic residues that do not participate in hydrolysis, such as methylphenoxy groups or other groups shown in the scientific paper of Allcock,et al., Macromolecule 10:824 (1977). Methods of synthesis of thehydrogel materials, as well as methods for preparing such hydrogels, areknown in the art.

Other components may also be included in the formulation, including butnot limited to any of the following: (1) buffers to provide appropriatepH and isotonicity; (2) lubricants; (3) viscous materials to retain thecells at or near the site of administration, including, for example,alginates, agars and plant gums; and (4) other cell types that mayproduce a desired effect at the site of administration, such as, forexample, enhancement or modification of the formation of tissue or itsphysicochemical characteristics, or as support for the viability of thecells, or inhibition of inflammation or rejection. The cells may becovered by an appropriate wound covering to prevent cells from leavingthe site. Such wound coverings are known as those of skill in the art.

Formulation of a Cartilage or Bone Tissue Patch

Culture or co-cultures of PPDCs in a pre-shaped well enables themanufacture of a tissue patch of pre-determined thickness and volume.The volume of the resulting tissue patch is dependent upon the volume ofthe well and upon the number of PPDCs in the well. Tissue of optimalpre-determined volume may be prepared by routine experimentation byaltering either or both of the aforementioned parameters.

The cell contacting surface of the well may be coated with a moleculethat discourages adhesion of PPDCs to the cell contacting surface.Preferred coating reagents include silicon based reagents i.e.,dichlorodimethylsilane or polytetrafluoroethylene based reagents, i.e.,TEFLON. Procedures for coating materials with silicon based reagents,specifically dichlorodimethylsilane, are well known in the art. See forexample, Sambrook et al. (1989) “Molecular Cloning A Laboratory Manual”,Cold Spring Harbor Laboratory Press, the disclosure of which isincorporated by reference herein. It is appreciated that otherbiocompatible reagents that prevent the attachment of cells to thesurface of the well may be useful in the practice of the instantinvention.

Alternatively, the well may be cast from a pliable or moldablebiocompatible material that does not permit attachment of cells per se.Preferred materials that prevent such cell attachment include, but arenot limited to, agarose, glass, untreated cell culture plastic andpolytetrafluoroethylene, i.e., TEFLON. Untreated cell culture plastics,i.e., plastics that have not been treated with or made from materialsthat have an electrostatic charge are commercially available, and may bepurchased, for example, from Falcon Labware, Becton-Dickinson, LincolnPark, N.J. The aforementioned materials, however, are not meant to belimiting. It is appreciated that any other pliable or moldablebiocompatible material that inherently discourages the attachment ofPPDCs may be useful in the practice of the instant invention.

The size and shape of the well may be determined by the size and shapeof the tissue defect to be repaired. For example, it is contemplatedthat the well may have a cross-sectional surface area of 25 cm². This isthe average cross-sectional surface area of an adult, human femoralchondyle. Accordingly, it is anticipated that a single piece ofcartilage may be prepared in accordance with the invention in order toresurface the entire femoral chondyle. The depth of the well ispreferably greater than about 0.3 cm and preferably about 0.6 cm indepth. The thickness of natural articular cartilage in an adultarticulating joint is usually about 0.3 cm. Accordingly, the depth ofthe well should be large enough to permit a cartilage patch of about 0.3cm to form. The well should be deep enough to contain culture mediumoverlaying the tissue patch.

It is contemplated that a tissue patch prepared in accordance with theinvention may be “trimmed” to a pre-selected size and shape by a surgeonperforming surgical repair of the damaged tissue. Trimming may beperformed with the use of a sharp cutting implement, i.e., a scalpel, apair of scissors or an arthroscopic device fitted with a cutting edge,using procedures well known in the art.

The pre-shaped well may be cast in a block of agarose gel under asepticconditions. Agarose is an economical, biocompatible, pliable andmoldable material that can be used to cast pre-shaped wells, quickly andeasily. As mentioned above, the dimensions of the well may dependentupon the size of the resulting tissue plug that is desired.

A pre-shaped well may be prepared by pouring a hot solution of molten LTagarose (BioRad, Richmond, Calif.) into a tissue culture dish containinga cylinder, the cylinder having dimensions that mirror the shape of thewell to be formed. The size and shape of the well may be chosen by theartisan and may be dependent upon the shape of the tissue defect to berepaired. Once the agarose has cooled and solidified around thecylinder, the cylinder is carefully removed with forceps. The surface ofthe tissue culture dish that is exposed by the removal of the cylinderis covered with molten agarose. This seals the bottom of the well andprovides a cell adhesive surface at the base of the well. When the newlyadded molten LT agarose cools and solidifies, the resulting pre-shapedwell is suitable for culturing, and inducing the differentiation ofPPDCs. It is appreciated, however, that alternative methods may be usedto prepare a pre-shaped well useful in the practice of the invention.

PPDCs in suspension may be seeded into and cultured in the pre-shapedwell. The PPDCs may be induced to differentiate to a chondrogenic orosteogenic phenotype in culture in the well or may have been induced todifferentiate prior to seeding in the well. The cells may be diluted bythe addition of culture medium to a cell density of about 1×10⁵ to 1×10⁹PPDCs per milliliter.

The cells may form a cohesive plug of cells. The cohesive plug of cellsmay be removed from the well and surgically implanted into the tissuedefect. It is anticipated that undifferentiated PPDCs may differentiatein situ thereby to form tissue in vivo.

Cartilage and bone defects may be identified inferentially by usingcomputer aided tomography (CAT scanning); X-ray examination, magneticresonance imaging (MRI), analysis of synovial fluid or serum markers orby any other procedures known in the art. Defects in mammals also arereadily identifiable visually during arthroscopic examination or duringopen surgery of the joint. Treatment of the defects can be effectedduring an arthroscopic or open surgical procedure using the methods andcompositions disclosed herein.

Accordingly, once the defect has been identified, the defect may betreated by the following steps of (1) surgically implanting at thepre-determined site a tissue patch prepared by the methodologiesdescribed herein, and (2) permitting the tissue patch to integrate intopre-determined site.

The tissue patch optimally has a size and shape such that when the patchis implanted into the defect, the edges of the implanted tissue contactdirectly the edges of the defect. In addition, the tissue patch may befixed in place during the surgical procedure. This can be effected bysurgically fixing the patch into the defect with biodegradable suturesand/or by applying a bioadhesive to the region interfacing the patch andthe defect.

In some instances, damaged tissue maybe surgically excised prior the toimplantation of the patch of tissue.

Transplantation of PPDCs Using Scaffolds

The cells of the invention or co-cultures thereof may be seeded onto orinto a three-dimensional scaffold and implanted in vivo, where theseeded cells will proliferate on the framework and form a replacementcartilage or bone tissue in vivo in cooperation with the cells of thepatient.

In some aspects of the invention, the matrix comprises decellularizedtissue, such as extracellular matrix, cell lysates (e.g., soluble cellfractions), or combinations thereof, of the PPDCs. In some embodiments,the matrix is biodegradable. In some aspects of the invention, thematrix comprises natural or synthetic polymers. Matrices of theinvention include biocompatible scaffolds, lattices, self-assemblingstructures and the like, whether biodegradable or not, liquid or solid.Such matrices are known in the arts of cell-based therapy, surgicalrepair, tissue engineering, and wound healing. Preferably the matricesare pretreated (e.g., seeded, inoculated, contacted with) with thecells, extracellular matrix, conditioned medium, cell lysate, orcombination thereof, of the invention. More preferably the matrices arepopulated with cells in close association to the matrix or its spaces.In some aspects of the invention, the cells adhere to the matrix. Insome embodiments, the cells are contained within or bridge interstitialspaces of the matrix. Most preferred are those seeded matrices whereinthe cells are in close association with the matrix and which, when usedtherapeutically, induce or support ingrowth of the patient's cellsand/or proper angiogenesis. The seeded matrices can be introduced into apatient's body in any way known in the art, including but not limited toimplantation, injection, surgical attachment, transplantation with othertissue, injection, and the like. The matrices of the invention may beconfigured to the shape and/or size of a tissue or organ in vivo.

For example, but not by way of limitation, the scaffold may be designedsuch that the scaffold structure: (1) supports the seeded cells withoutsubsequent degradation; (2) supports the cells from the time of seedinguntil the tissue transplant is remodeled by the host tissue; (2) allowsthe seeded cells to attach, proliferate, and develop into a tissuestructure having sufficient mechanical integrity to support itself invitro, at which point, the scaffold is degraded. A review of scaffolddesign is provided by Hutmacher, J. Biomat. Sci. Polymer Edn.,12(1):107-124 (2001).

Scaffolds of the invention can be administered in combination with anyone or more growth factors, cells, for example stem cells, bone marrowcells, chondrocytes, chondroblasts, osteocytes, osteoblasts,osteoclasts, bone lining cells, or their precursors, drugs or othercomponents described above that stimulate tissue formation or otherwiseenhance or improve the practice of the invention. The PPDCs to be seededonto the scaffolds may be genetically engineered to express growthfactors or drugs.

The cells of the invention can be used to produce new tissue in vitro,which can then be implanted, transplanted or otherwise inserted into asite requiring tissue repair, replacement or augmentation in a patient.

In a non-limiting embodiment, the cells of the invention are used toproduce a three-dimensional tissue construct in vitro, which is thenimplanted in vivo. As an example of the production of three-dimensionaltissue constructs, see U.S. Pat. No. 4,963,489, which is incorporatedherein by reference. For example, the cells of the invention may beinoculated or “seeded” onto a three-dimensional framework or scaffold,and proliferated or grown in vitro to form a living tissue that can beimplanted in vivo.

The cells of the invention can be grown freely in a culture vessel tosub-confluency or confluency, lifted from the culture and inoculatedonto a three-dimensional framework. Inoculation of the three-dimensionalframework with a high concentration of cells, e.g., approximately 10⁶ to5×10⁷ cells per milliliter, will result in the establishment of thethree-dimensional support in relatively shorter periods of time.

Examples of scaffolds which may be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe tradename VICRYL (Ethicon, Inc., Somerville, N.J.). Foams, composedof, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699, are also possiblescaffolds. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. These materials are frequently used as supports for growthof tissue.

The three-dimensional framework may be made of ceramic materialsincluding, but not limited to: mono-, di-, tri-, alpha-tri-, beta-tri-,and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS (University of Florida, Gainesville, Fla.), and mixturesthereof. There are a number of suitable porous biocompatible ceramicmaterials currently available on the commercial market such as SURGIBON(Unilab Surgibone, Inc., Canada), ENDOBON (Merck Biomaterial France,France), CEROS (Mathys, A. G., Bettlach, Switzerland), and INTERPORE(Interpore, Irvine, Calif., United States), and mineralized collagenbone grafting products such as HEALOS (Orquest, Inc., Mountain View,Calif.) and VITOSS, RHAKOSS, and CORTOSS (Orthovita, Malvern, Pa.). Theframework may be a mixture, blend or composite of natural and/orsynthetic materials. In some embodiments, the scaffold is in the form ofa cage. In a preferred embodiment, the scaffold is coated with collagen.

According to a preferred embodiment, the framework is a felt, which canbe composed of a multifilament yarn made from a bioabsorbable material,e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarnis made into a felt using standard textile processing techniquesconsisting of crimping, cutting, carding and needling.

In another preferred embodiment the cells of the invention are seededonto foam scaffolds that may be composite structures. In addition, thethree-dimensional framework may be molded into a useful shape, such asthat of the external portion of the ear, a bone, joint or other specificstructure in the body to be repaired, replaced or augmented.

In another preferred embodiment, the cells of the invention are seededonto a framework comprising a prosthetic device for implantation into apatient, as described in U.S. Pat. No. 6,200,606, incorporated herein byreference. As described therein, a variety of clinically usefulprosthetic devices have been developed for use in bone and cartilagegrafting procedures. (see e.g. Bone Grafts and Bone Substitutions. Ed.M. B. Habal & A. H. Reddi, W.B. Saunders Co., 1992). For example,effective knee and hip replacement devices have been and continue to bewidely used in the clinical environment. Many of these devices arefabricated using a variety of inorganic materials having low immunogenicactivity, which safely function in the body. Examples of syntheticmaterials which have been tried and proven include titanium alloys,calcium phosphate, ceramic hydroxyapatite, and a variety of stainlesssteel and cobalt-chrome alloys. These materials provide structuralsupport and can form a scaffolding into which host vascularization andcell migration can occur. The present invention provides a source ofcells which may be used to “seed” such prosthetic devices. In thepreferred embodiment PPDCs are first mixed with a carrier materialbefore application to a device. Suitable carriers well known to thoseskilled in the art include, but are not limited to, gelatin, fibrin,collagen, starch, polysaccharides, saccharides, proteoglycans, syntheticpolymers, calcium phosphate, or ceramics.

The framework may be treated prior to inoculation of the cells of theinvention in order to enhance cell attachment. For example, prior toinoculation with the cells of the invention, nylon matrices could betreated with 0.1 molar acetic acid and incubated in polylysine, PBS,and/or collagen to coat the nylon. Polystyrene could be similarlytreated using sulfuric acid.

In addition, the external surfaces of the three-dimensional frameworkmay be modified to improve the attachment or growth of cells anddifferentiation of tissue, such as by plasma coating the framework oraddition of one or more proteins (e.g., collagens, elastic fibers,reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, amongothers.

In some embodiments, the scaffold is comprised of or is treated withmaterials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as ePTFE, and segmented polyurethaneureasilicones, such as PURSPAN (The Polymer Technology Group, Inc.,Berkeley, Calif.). These materials can be further treated to render thescaffold non-thrombogenic. Such treatments include anti-thromboticagents such as heparin, and treatments which alter the surface charge ofthe material such as plasma coating.

In some embodiments, the surface of the scaffold is textured. Forexample, in some aspects of the invention, the scaffold is provided witha groove and ridge pattern. The grooves are preferably less than about500 microns, more preferably less than about 100 microns, and mostpreferably between about 10 nanometers and 10 microns. Such“microgrooves” allow the cells to align and/or migrate guided by thesurface grooves. See, e.g., Odontology. 2001; 89(1):2-11. The texturedscaffold may be used, for example, as a dental implant.

In some embodiments, it is important to re-create in culture thecellular microenvironment found in vivo, such that the extent to whichthe cells of the invention are grown prior to implantation in vivo oruse in vitro may vary. In addition, growth factors, chondrogenicdifferentiation inducing agents, osteogenic inducing agents, andangiogenic factors may be added to the culture medium prior to, during,or subsequent to inoculation of the cells to trigger differentiation andtissue formation by the PPDCs or co-cultures thereof.

The three-dimensional framework may be modified so that the growth ofcells and the production of tissue thereon is enhanced, or so that therisk of rejection of the implant is reduced. Thus, one or morebiologically active compounds, including, but not limited to,anti-inflammatories, immunosuppressants or growth factors, may be addedto the framework.

Therapeutic Uses for Extracellular Matrix or Cell Lysate Derived fromPPDCs

As an alternative to implanting the cells of the invention, or livingtissue produced therefrom, a subject in need of tissue repair,replacement, or augmentation may benefit from the administration of acomponent or product of PPDCs, such as the extracellular matrix (ECM) orcell lysate produced by those cells.

In some embodiments, after the cells of the invention have been culturedin vitro, such as, for example, by using a three-dimensional scaffoldsystem described herein, such that a desired amount of ECM has beensecreted onto the framework. Once ECM is secreted onto the framework,the cells may be removed. The ECM may be processed for further use, forexample, as an injectable preparation.

In some embodiments, the cells are killed and cellular debris (e.g.,cellular membranes) is removed from the framework. This process may becarried out in a number of different ways. For example, the livingtissue can be flash-frozen in liquid nitrogen without acryopreservative, or the tissue can be immersed in sterile distilledwater so that the cells burst in response to osmotic pressure. Once thecells have been killed, the cellular membranes may be disrupted andcellular debris removed by treatment with a mild detergent rinse, suchas EDTA, CHAPS or a zwitterionic detergent. An advantage to using a milddetergent rinse is that it solubilizes membrane-bound proteins, whichare often highly antigenic.

Alternatively, the tissue can be enzymatically digested and/or extractedwith reagents that break down cellular membranes. Example of suchenzymes include, but are not limited to, hyaluronidase, dispase,proteases, and nucleases (for example, deoxyribonuclease andribonuclease). Examples of detergents include non-ionic detergents suchas, for example, alkylaryl polyether alcohol (TRITON® X-100),octylphenoxy polyethoxy-ethanol (Rohm and Haas Philadelphia, Pa.),BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co., SanDiego, Calif.), polysorbate 20 (TWEEN 20®), a polyethoxyethanol sorbitanmonolaureate (Rohm and Haas), polyethylene lauryl ether (Rohm and Haas);and ionic detergents such as, for example, sodium dodecyl sulphate,sulfated higher aliphatic alcohols, sulfonated alkanes and sulfonatedalkylarenes containing 7 to 22 carbon atoms in a branched or unbranchedchain.

Scaffolds comprising the ECm may be used therapeutically as describedabove. Alternatively, ECM may be collected from the scaffold. Thecollection of the ECM can be accomplished in a variety of ways,depending, for example, on whether the scaffold is biodegradable ornon-biodegradable. For example, if the framework is non-biodegradable,the ECM can be removed by subjecting the framework to sonication, highpressure water jets, mechanical scraping, or mild treatment withdetergents or enzymes, or any combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM or cell lysate produced by the cells of the invention.

Once the ECM has been collected, it may be processed further. The ECMcan be homogenized to fine particles using techniques well known in theart such as, for example, by sonication, so that they can pass through asurgical needle. ECM components can be crosslinked, if desired, by gammairradiation. Preferably, the ECM can be irradiated between 0.25 to 2mega rads to sterilize and crosslink the ECM. Chemical crosslinkingusing agents that are toxic, such as glutaraldehyde, is possible but notgenerally preferred.

Cell lysates prepared from the populations of the postpartum-derivedcells also have many utilities. In one embodiment, whole cell lysatesare prepared, e.g., by disrupting cells without subsequent separation ofcell fractions. In another embodiment, a cell membrane fraction isseparated from a soluble fraction of the cells by routine methods knownin the art, e.g., centrifugation, filtration, or similar methods. Use ofsoluble cell fractions in vivo allows the beneficial intracellularmilieu to be used in a patient without triggering rejection or anadverse response. Methods of lysing cells are well-known in the art andinclude various means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their growth medium and thus containingsecreted growth factors and the like, or may be prepared from cellswashed free of medium in, for example, PBS or other solution. Washedcells may be resuspended at concentrations greater than the originalpopulation density if preferred. Cell lysates prepared from populationsof postpartum-derived cells may be used as is, further concentrated, byfor example, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Cell lysates may be used in vitro or in vivo, alone or for example, withcells. The cell lysates, if introduced in vivo, may be introducedlocally at a site of treatment, or remotely to provide, for exampleneeded cellular growth factors to a patient.

The amounts and/or ratios of proteins may be adjusted by mixing the ECMor cell lysate produced by the cells of the invention with ECM or celllysate of one or more other cell types. In addition, biologically activesubstances such as proteins, growth factors and/or drugs, can beincorporated into the ECM or cell lysate preparation. Exemplarybiologically active substances include anti-inflammatory agents andgrowth factors which promote healing and tissue repair. Cells may beco-administered with the ECM or cell lysates of the invention. ECM orcell lysate of PPDCs may be formulated for administration as describedabove for PPDCs.

The above described process for preparing injectable ECM or cell lysateis preferably carried out under sterile conditions using sterilematerials. The processed ECM or cell lysate in a pharmaceuticallyacceptable carrier can be injected intradermally or subcutaneously totreat bone or cartilage conditions, for example, by augmenting tissue orrepairing or correcting congenital anomalies, acquired defects orcosmetic defects.

Use of PPDCs for In Vitro Screening of Drug Efficacy or Toxicity

The cells and tissues of the invention may be used in vitro to screen awide variety of compounds for effectiveness and cytotoxicity ofpharmaceutical agents, growth/regulatory factors, anti-inflammatoryagents. To this end, the cells of the invention, or tissue culturesdescribed above, are maintained in vitro and exposed to the compound tobe tested. The activity of a cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth/regulatory factorsmay be assessed by analyzing the number of living cells in vitro, e.g.,by total cell counts, and differential cell counts. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens. The effect of various drugson the cells of the invention either in suspension culture or in thethree-dimensional system described above may be assessed.

The cells and tissues of the invention may be used as model systems forthe study of physiological or pathological conditions. For example,joints that are immobilized suffer relatively quickly in a number ofrespects. The metabolic activity of chondrocytes appears affected asloss of proteoglycans and an increase in water content are soonobserved. The normal white, glistening appearance of the cartilagechanges to a dull, bluish color, and the cartilage thickness is reduced.However, the amount of this change that is due to nutritional deficiencyversus the amount due to upset in the stress-dependent metabolichomeostasis is not yet clear. The cells and tissues of the invention maybe used to determine the nutritional requirements of cartilage underdifferent physical conditions, e.g., intermittent pressurization, and bypumping action of nutrient medium into and out of the cartilageconstruct. This may be especially useful in studying underlying causesfor age-related or injury-related decrease in tensile strength of, forexample, articular cartilage, e.g., in the knee, that predispose theweakened cartilage to traumatic damage.

The cells and tissues of the invention may also be used to study themechanism of action of cytokines, growth factors and inflammatorymediators, e.g., IL-1, TNF and prostaglandins. In addition, cytotoxicand/or pharmaceutical agents can be screened for those that are mostefficacious for a particular patient, such as those that reduce orprevent resorption of cartilage or bone otherwise enhance the balancedgrowth thereof. Agents that prove to be efficacious in vitro could thenbe used to treat the patient therapeutically.

Use of PPDCs to Produce Biological Molecules

In a further embodiment, the cells of the invention can be cultured invitro to produce biological products in high yield. For example, suchcells, which either naturally produce a particular biological product ofinterest (e.g., a growth factor, regulatory factor, or peptide hormone),or have been genetically engineered to produce a biological product,could be clonally expanded using, for example, the three-dimensionalculture system described above. If the cells excrete the biologicalproduct into the nutrient medium, the product can be readily isolatedfrom the spent or conditioned medium using standard separationtechniques, e.g., such as differential protein precipitation,ion-exchange chromatography, gel filtration chromatography,electrophoresis, and high performance liquid chromatography. A“bioreactor” may be used to take advantage of the flow method forfeeding, for example, a three-dimensional culture in vitro.

Essentially, as fresh media is passed through the three-dimensionalculture, the biological product is washed out of the culture and maythen be isolated from the outflow, as above.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells be lysed. Thebiological product may then be purified using any one or more of theabove-listed techniques.

Kits

The PPDCs and components and products thereof can conveniently beemployed as part of a kit, for example, for culture or implantation.Accordingly, the invention provides a kit including the PPDCs andadditional components, such as a matrix (e.g., a scaffold), hydratingagents (e.g., physiologically-compatible saline solutions, prepared cellculture media), cell culture substrates (e.g., culture dishes, plates,vials, etc.), cell culture media (whether in liquid or powdered form),antibiotic compounds, hormones, and the like. While the kit can includeany such components, preferably it includes all ingredients necessaryfor its intended use. If desired, the kit also can include cells(typically cryopreserved), which can be seeded into the lattice asdescribed herein.

In another aspect, the invention provides kits that utilize the PPDCs,PPDC populations, components and products of PPDCs in various methodsfor augmentation, regeneration, and repair as described above. In someembodiments, the kits may include one or more cell populations,including at least PPDCs and a pharmaceutically acceptable carrier(liquid, semi-solid or solid). The kits also optionally may include ameans of administering the cells, for example by injection. The kitsfurther may include instructions for use of the cells. Kits prepared forfield hospital use, such as for military use, may include full-proceduresupplies including tissue scaffolds, surgical sutures, and the like,where the cells are to be used in conjunction with repair of acuteinjuries. Kits for assays and in vitro methods as described herein maycontain one or more of (1) PPDCs or components or products of PPDCs, (2)reagents for practicing the in vitro method, (3) other cells or cellpopulations, as appropriate, and (4) instructions for conducting the invitro method.

Cryopreservation and Banking PPDCs

PDCs of the invention can be cryopreserved and maintained or stored in a“cell bank”. Cryopreservation of cells of the invention may be carriedout according to known methods. For example, but not by way oflimitation, cells may be suspended in a “freeze medium” such as, forexample, culture medium further comprising 0 to 95 percent FBS and 0 to10 percent dimethylsulfoxide (DMSO), with or without 5 to 10 percentglycerol, at a density, for example, of about 0.5 to 10×10⁶ cells permilliliter. The cryopreservation medium may comprise cryopreservationagents including but not limited to methylcellulose. The cells aredispensed into glass or plastic ampoules that are then sealed andtransferred to the freezing chamber of a controlled rate freezer. Theoptimal rate of freezing may be determined empirically. A programmablerate freezer for example, can give a change in temperature of −1 to −10°C. per minute. The preferred cryopreservation temperature is about −80°C. to about −180° C., more preferably is about −90° C. to about −160°C., and most preferably is about −125 to about −140° C. Cryopreservedcells preferably are transferred to liquid nitrogen prior to thawing foruse. In some embodiments, for example, once the ampoules have reachedabout −90° C., they are transferred to a liquid nitrogen storage area.Cryopreserved cells can be stored for a period of years.

The cryopreserved cells of the invention constitute a bank of cells,portions of which can be “withdrawn” by thawing and then used as needed.Thawing should generally be carried out rapidly, for example, bytransferring an ampoule from liquid nitrogen to a 37° C. water bath. Thethawed contents of the ampoule should be immediately transferred understerile conditions to a culture vessel containing an appropriate mediumsuch as DMEM conditioned with 10 percent FBS.

In yet another aspect, the invention also provides for banking oftissues, cells, cellular components and cell populations of theinvention. As discussed above, the cells are readily cryopreserved. Theinvention therefore provides methods of cryopreserving the cells in abank, wherein the cells are stored frozen and associated with a completecharacterization of the cells based on immunological, biochemical andgenetic properties of the cells. The cells so frozen can be used forautologous, syngeneic, or allogeneic therapy, depending on therequirements of the procedure and the needs of the patient. Preferably,the information on each cryopreserved sample is stored in a computer,which is searchable based on the requirements of the surgeon, procedureand patient with suitable matches being made based on thecharacterization of the cells or populations. Preferably, the cells ofthe invention are grown and expanded to the desired quantity of cellsand therapeutic cell compositions are prepared either separately or asco-cultures, in the presence or absence of a matrix or support. Whilefor some applications it may be preferable to use cells freshlyprepared, the remainder can be cryopreserved and banked by freezing thecells and entering the information in the computer to associate thecomputer entry with the samples. Even where it is not necessary to matcha source or donor with a recipient of such cells, for immunologicalpurposes, the bank system makes it easy to match, for example, desirablebiochemical or genetic properties of the banked cells to the therapeuticneeds. Upon matching of the desired properties with a banked sample, thesample is retrieved, and readied for therapeutic use. Cell lysates orcomponents prepared as described herein may also be preserved (e.g.,cryopreserved, lyophilized) and banked in accordance with the presentinvention.

The following examples describe several aspects of embodiments of theinvention in greater detail. These examples are provided to furtherillustrate, not to limit, aspects of the invention described herein.

EXAMPLES Example 1 Derivation of Cells from Postpartum Tissues

The objective of this study was to derive populations of cells fromplacental and umbilical cord tissues. Postpartum umbilical cord andplacenta were obtained upon birth of either a full term or pre-termpregnancy. Cells were harvested from 5 separate donors of umbilical cordand placental tissue. Different methods of cell isolation were testedfor their ability to yield cells with: 1) the potential to differentiateinto cells with different phenotypes, or 2) the potential to providecritical trophic factors useful for other cells and tissues.

Methods & Materials

Umbilical cord cell derivation. Umbilical cords were obtained fromNational Disease Research Interchange (NDRI, Philadelphia, Pa.). Thetissues were obtained following normal deliveries. The cell isolationprotocol was performed aseptically in a laminar flow hood. To removeblood and debris, the umbilical cord was washed in phosphate bufferedsaline (PBS; Invitrogen, Carlsbad, Calif.) in the presence ofantimycotic and antibiotic (100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B (Invitrogen Carlsbad, Calif.)). The tissues were thenmechanically dissociated in 150 cm² tissue culture plates in thepresence of 50 milliliters of medium (DMEM-Low glucose or DMEM-Highglucose; Invitrogen) until the tissue was minced into a fine pulp. Thechopped tissues were transferred to 50 milliliter conical tubes(approximately 5 grams of tissue per tube). The tissue was then digestedin either DMEM-Low glucose medium or DMEM-High glucose medium, eachcontaining antimycotic and antibiotic (100 Units/milliliter penicillin,100 micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B (Invitrogen)) and digestion enzymes. In some experiments,an enzyme mixture of collagenase and dispase was used (“C:D;”collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; and dispase(Invitrogen), 50 Units/milliliter in DMEM-Low glucose medium). In otherexperiments a mixture of collagenase, dispase and hyaluronidase(“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter, inDMEM-Low glucose). The conical tubes containing the tissue, medium anddigestion enzymes were incubated at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,and the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth medium (DMEM-Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 100Units/milliliter of penicillin, 100 microgram/milliliter streptomycin,0.25 microgram/milliliter amphotericin B (Invitrogen, Carlsbad, Calif.).The cell suspension was filtered through a 70-micrometer nylon cellstrainer (BD Biosciences). An additional 5 milliliter rinse comprisingGrowth medium was passed through the strainer. The cell suspension wasthen passed through a 40-micrometer nylon cell strainer (BD Biosciences)and chased with a rinse of an additional 5 milliliters of Growth medium.

The filtrate was resuspended in Growth medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated, and the cells were resuspended in 50 milliliters of freshGrowth medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh Growth medium. Thenumber of viable cells was determined using Trypan Blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cord cells were seeded at 5,000cells/cm² onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning,N.Y.) in Growth medium (DMEM-Low glucose (Invitrogen), 15 percent (v/v)defined bovine serum (Hyclone, Logan, Utah; Lot#AND18475), 0.001 percent(v/v) 2-mercaptoethanol (Sigma), 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B (Invitrogen)). After about 2-4 days, spent medium wasaspirated from the flasks. Cells were washed with PBS three times toremove debris and blood-derived cells. Cells were then replenished withGrowth medium and allowed to grow to confluence (about 10 days frompassage 0 to passage 1). On subsequent passages (from passage 1 to 2,etc.), cells reached sub-confluence (75-85 percent confluence) in 4-5days. For these subsequent passages, cells were seeded at 5000cells/cm². Cells were grown in a humidified incubator with 5 percentcarbon dioxide and 20 percent oxygen at 37° C.

Placental Cell Isolation. Placental tissue was obtained from NDRI(Philadelphia, Pa.). The tissues were from a pregnancy and were obtainedat the time of a normal surgical delivery. Placental cells were isolatedas described for umbilical cord cell isolation.

The following example applies to the isolation of separate populationsof maternal-derived and neonatal-derived cells from placental tissue.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (100 U/milliliter penicillin, 100 microgram/milliliterstreptomycin, 0.25 microgram/milliliter amphotericin B; Invitrogen) toremove blood and debris. The placental tissue was then dissected intothree sections: top-line (neonatal side or aspect), mid-line (mixed cellisolation neonatal and maternal or villous region), and bottom line(maternal side or aspect).

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. Eachsection was then mechanically dissociated in 150 cm² tissue cultureplates in the presence of 50 milliliters of DMEM-Low glucose(Invitrogen) to a fine pulp. The pulp was transferred to 50 milliliterconical tubes. Each tube contained approximately 5 grams of tissue. Thetissue was digested in either DMEM-Low glucose or DMEM-High glucosemedium containing antimycotic and antibiotic (100 Units/milliliterpenicillin, 100 micrograms/milliliter streptomycin, 0.25micrograms/milliliter amphotericin B (Invitrogen)) and digestionenzymes. In some experiments an enzyme mixture of collagenase anddispase (“C:D”) was used containing collagenase (Sigma, St Louis, Mo.)at 500 Units/milliliter and dispase (Invitrogen) at 50Units/milliliterin DMEM-Low glucose medium. In other experiments amixture of collagenase, dispase, and hyaluronidase (C:D:H) was used(collagenase, 500 Units/milliliter; dispase, 50 Units/milliliter; andhyaluronidase (Sigma), 5 Units/milliliter in DMEM-Low glucose). Theconical tubes containing the tissue, medium, and digestion enzymes wereincubated for 2 h at 37° C. in an orbital shaker (Environ, Brooklyn,N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the resultant supernatant was aspirated off. The pellet was resuspendedin 20 milliliter of Growth medium (DMEM-Low glucose (Invitrogen), 15%(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis,Mo.), antibiotic/antimycotic (100 U/milliliter penicillin, 100microgram/milliliter streptomycin, 0.25 microgram/milliliteramphotericin B; Invitrogen)). The cell suspension was filtered through a70 micrometer nylon cell strainer (BD Biosciences), chased by a rinsewith an additional 5 milliliters of Growth medium. The total cellsuspension was passed through a 40 micrometer nylon cell strainer (BDBiosciences) followed with an additional 5 milliliters of Growth mediumas a rinse.

The filtrate was resuspended in Growth medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cell pellet was resuspended in 50 milliliters of freshGrowth medium. This process was repeated twice more. After the finalcentrifugation, supernatant was aspirated and the cell pellet wasresuspended in 5 milliliters of fresh Growth medium. A cell count wasdetermined using the Trypan Blue Exclusion test. Cells were thencultured at standard conditions.

LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) Cell Isolation.Cells were isolated from umbilical cord in DMEM-Low glucose medium withLIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) (2.5 milligramsper milliliter, Blendzyme 3; Roche Applied Sciences, Indianapolis, Ind.)and hyaluronidase (5 Units/milliliter, Sigma). Digestion of the tissueand isolation of the cells was as described for other proteasedigestions above using a LIBERASE (Boehringer Mannheim Corp.,Indianapolis, Ind.)/hyaluronidase mixture in place of the C:D or C:D:Henzyme mixture. Tissue digestion with LIBERASE (Boehringer MannheimCorp., Indianapolis, Ind.) resulted in the isolation of cell populationsfrom postpartum tissues that expanded readily.

Cell isolation using other enzyme combinations. Procedures were comparedfor isolating cells from the umbilical cord using differing enzymecombinations. Enzymes compared for digestion included: i) collagenase;ii) dispase; iii) hyaluronidase; iv) collagenase:dispase mixture (C;D);v) collagenase:hyaluronidase mixture (C:H); vi) dispase:hyaluronidasemixture (D:H); and vii) collagenase:dispase:hyaluronidase mixture(C:D:H). Differences in cell isolation utilizing these different enzymedigestion conditions were observed (Table 1-1).

Isolation of cells from residual blood in the cords. Attempts were madeto isolate pools of cells from umbilical cord by different approaches.In one instance umbilical cord was sliced and washed with Growth mediumto dislodge the blood clots and gelatinous material. The mixture ofblood, gelatinous material, and Growth medium was collected andcentrifuged at 150×g. The pellet was resuspended and seeded ontogelatin-coated flasks in Growth medium. From these experiments a cellpopulation was isolated that readily expanded.

Isolation of cells from Cord Blood. Cells have also been isolated fromcord blood samples attained from NDRI. The isolation protocol used herewas that of International Patent Application WO02/29971 by Ho et al.Samples (50 milliliter and 10.5 milliliters, respectively) of umbilicalcord blood (NDRI, Philadelphia Pa.) were mixed with lysis buffer(filter-sterilized 155 millimolar ammonium chloride, 10 millimolarpotassium bicarbonate, 0.1 millimolar EDTA buffered to pH 7.2 (allcomponents from Sigma, St. Louis, Mo.)). Cells were lysed at a ratio of1:20 cord blood to lysis buffer. The resulting cell suspension wasvortexed for 5 seconds, and incubated for 2 minutes at ambienttemperature. The soluble cell fraction was centrifuged (10 minutes at200×g). The cell pellet was resuspended in complete minimal essentialmedium (Gibco, Carlsbad Calif.) containing 10 percent fetal bovine serum(Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech Herndon, Va.),100 Units penicillin per 100 milliliters and 100 micrograms streptomycinper 100 milliliters (Gibco, Carlsbad, Calif.). The resuspended cellswere centrifuged (10 minutes at 200×g), the supernatant was aspirated,and the cell pellet was washed in complete medium. Cells were seededdirectly into either T75 flasks (Corning, N.Y.), T75 laminin-coatedflasks, or T175 fibronectin-coated flasks (both Becton Dickinson,Bedford, Mass.).

Isolation of postpartum-derived cells using different enzymecombinations and growth conditions. To determine whether cellpopulations can be isolated under different conditions and expandedunder a variety of conditions immediately after isolation, cells weredigested in Growth medium with or without 0.001 percent (v/v)2-mercaptoethanol (Sigma, St. Louis, Mo.), using the enzyme combinationof C:D:H, according to the procedures provided above. Placenta-derivedcells so isolated were seeded under a variety of conditions. All cellswere grown in the presence of penicillin/streptomycin. (Table 1-2).

Isolation of postpartum-derived cells using different enzymecombinations and growth conditions. In all conditions, cells attachedand expanded well between passage 0 and 1 (Table 1-2). Cells inconditions 5 to 8 and 13 to 16 were demonstrated to proliferate well upto 4 passages after seeding at which point they were cryopreserved. Allcells were banked.

Results

Cell isolation using different enzyme combinations. The combination ofC:D:H provided the best cell yield following isolation and generatedcells which expanded for many more generations in culture than the otherconditions (Table 1-1). An expandable cell population was not attainedusing collagenase or hyaluronidase alone. No attempt was made todetermine if this result is specific to the collagen that was tested.

Isolation of postpartum-derived cells using different enzymecombinations and growth conditions. Cells attached and expanded wellbetween passage 0 and 1 under all conditions tested for enzyme digestionand growth (Table 1-2). Cells in experimental conditions 5-8 and 13-16proliferated well up to 4 passages after seeding, at which point theywere cryopreserved. All cells were banked.

Isolation of cells from residual blood in the cords. Nucleated cellsattached and grew rapidly. These cells were analyzed by flow cytometryand were similar to cells obtained by enzyme digestion.

Isolation of cells from Cord Blood. The preparations contained red bloodcells and platelets. No nucleated cells attached and divided during thefirst 3 weeks. The medium was changed 3 weeks after seeding and no cellswere observed to attach and grow.

Summary. Populations of cells can be isolated from umbilical cord andplacental tissue most efficiently using the enzyme combinationcollagenase (a matrix metalloprotease), dispase (neutral protease), andhyaluronidase (a mucolytic enzyme which breaks down hyaluronic acid).LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.), which is aBlendzyme, may also be used. In the present study Blendzyme 3 which iscollagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) wasalso used together with hyaluronidase to isolate cells. These cellsexpand readily over many passages when cultured in Growth medium ongelatin-coated plastic.

Postpartum-derived cells were isolated from residual blood in the cordsbut not from cord blood. The presence of cells in blood clots washedfrom the tissue that adhere and grow under the conditions used may bedue to cells being released during the dissection process. TABLE 1-1Isolation of cells from umbilical cord tissue using varying enzymecombinations Enzyme Digest Cells Isolated Cell Expansion Collagenase X XDispase     +(>10 h) + Hyaluronidase X X Collagenase:Dispase   ++(<3 h)++ Collagenase:Hyaluronidase   ++(<3 h) + Dispase:Hyaluronidase    +(>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++Key:+ = good,++ = very good,+++ = excellent,X = no success

TABLE 1-2 Isolation and culture expansion of postpartum-derived cellsunder varying conditions: Condition Medium 15% FBS BME Gelatin 20% O2Growth Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg YY N Y N 4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) YEGF/FGF (20 ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20ng/mL) 7 DMEM-Lg N (2%) Y N Y PDGF/VEGF (Fibronectin) 8 DMEM-Lg N (2%) YN N (5%) PDGF/VEGF (Fibronectin) 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N(5%) N 11 DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%)N N (Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N(5%) EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N Y PDGF/VEGF (Fibronectin)16 DMEM-Lg N (2%) N N N (5%) PDGF/VEGF (Fibronectin)

REFERENCE

-   1. HO, Tony, W.; KOPEN, Gene, C.; RIGHTER, William, F.; RUTKOWSKI,    J., Lynn; HERRING, W., Joseph; RAGAGLIA, Vanessa; WAGNER, Joseph    WO2003025149 A2 CELL POPULATIONS WHICH CO-EXPRESS CD49C AND CD90,    NEURONYX, INC. Application No. US0229971 US, Filed 20020920, A2    Published 20030327, A3 Published 20031218.

Example 2 Evaluation of Growth Media for Postpartum-Derived Cells

Several cell culture media were evaluated for their ability to supportthe growth of placenta-derived cells. The growth of placenta-derivedcells in normal (20%) and low (5%) oxygen was assessed after 3 daysusing the MTS calorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/wellin 96 well plates in Growth medium (DMEM-low glucose (Gibco, CarlsbadCalif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan,Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco)). After 8 hours, the medium was changed as described in Table2-1, and cells were incubated in normal (20%, v/v) or low (5%, v/v)oxygen at 37° C., 5% CO₂ for 48 hours. MTS was added to the culturemedium (CELLTITER 96 AQueous One Solution Cell Proliferation Assay,Promega, Madison, Wis.) for 3 hours and the absorbance measured at 490nanometers (Molecular Devices, Sunnyvale Calif.). TABLE 2-1 Culturemedium Added fetal bovine serum % Culture Medium Supplier (v/v) DMEM-lowglucose Gibco Carlsbad CA 0, 2, 10 DMEM-high glucose Gibco 0, 2, 10 RPMI1640 Mediatech, Inc. 0, 2, 10 Herndon, VA Cell gro-free (Serum-free,Mediatech, Inc. — Protein-free) Ham's F10 Mediatech, Inc. 0, 2, 10 MSCGM(complete with Cambrex, Walkersville, 0, 2, 10 serum) MD Complete-serumfree Mediatech, Inc. — w/albumin Growth medium NA — Ham's F12 Mediatech,Inc. 0, 2, 10 Iscove's Mediatech, Inc. 0, 2, 10 Basal Medium Eagle'sMediatech, Inc. 0, 2, 10 DMEM/F12 (1:1) Mediatech, Inc. 0, 2, 10

Results

Standard curves for the MTS assay established a linear correlationbetween an increase in absorbance and an increase in cell number. Theabsorbance values obtained were converted into estimated cell numbersand the change (%) relative to the initial seeding was calculated.

The Effect of Serum. The addition of serum to media at normal oxygenconditions resulted in a reproducible dose-dependent increase inabsorbance and thus the viable cell number. The addition of serum tocomplete MSCGM resulted in a dose-dependent decrease in absorbance. Inthe media without added serum, cells grew in Cellgro, Ham's F10, andDMEM.

The Effect of Oxygen. Reduced oxygen appeared to increase the growthrate of cells in Growth Medium, Ham's F10, and MSCGM.

In decreasing order of growth, the media resulting in the best growth ofthe cells were Growth medium>MSCGM>Iscove's+10% FBS=DMEM-HG+10%FBS=Ham's F12+10% FBS=RPMI 1640+10% FBS.

Summary. Postpartum-derived cells may be grown in a variety of culturemedia in normal or low oxygen. Short-term growth of placenta-derivedcells was determined in 12 basal media with 0, 2, and 10% (v/v) serum in5% or 20% O₂. In general placenta-derived cells did not grow inserum-free conditions with the exceptions of Ham's F10 and Cellgro-free,which are also protein-free. Growth in these serum-free media wasapproximately 25-33% of the maximal growth observed with Growth mediumcontaining 15% serum. This study demonstrates that placenta-derivedcells may be grown in serum-free conditions and that Growth medium isone of several media (10% serum in Iscove's, RPMI or Ham's F12 media)that can be used to grow placenta-derived cells.

The most promising serum-free media was CELLGRO-FREE, a serum andprotein-free medium without hormones or growth factors, which isdesigned for the growth of mammalian cells in vitro (Mediatech productinformation).

Complete-serum free medium also developed for serum-free culture was notas effective in supporting growth of the placenta-derived cells.Complete-serum free was developed by Mediatech, based on a 50/50 mix ofDMEM/F12 with smaller percentages of RPMI 1640 and McCoy's 5A. Thismedium also contains selected trace elements and high molecular weightcarbohydrates, extra vitamins, a non-animal protein source, and a smallamount of BSA (1 gram/liter). It does not contain any insulin,transferrin, cholesterol, or growth or attachment factors. It isbicarbonate buffered for use with 5% CO₂. Originally designed forhybridomas and suspension cell lines, it may be suitable for someanchorage dependent cell lines.

Example 3 Growth of Postpartum-Derived Cells in Medium ContainingD-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). The growth of postpartum-derived cells in medium containingD-valine in the absence of L-valine was evaluated.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9), and umbilicalcord-derived cells (P5) were seeded at 5×10³ cells/cm² in gelatin-coatedT75 flasks (Corning, Corning, N.Y.). After 24 hours the medium wasremoved and the cells were washed with phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) to remove residual medium. The medium wasreplaced with a Modified Growth medium (DMEM with D-valine (specialorder Gibco), 15% (v/v) dialyzed fetal bovine serum (Hyclone, Logan,Utah), 0.001% (v/v) betamercaptoethanol (Sigma), 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin (Gibco)).

Results

Placenta-derived, umbilical cord-derived, and fibroblast cells seeded inthe D-valine-containing medium did not proliferate, unlike cells seededin Growth medium containing dialyzed serum. Fibroblasts changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after 4 weeks.

Summary. Postpartum-derived cells require L-valine for cell growth andfor long-term viability. L-valine is preferably not removed from thegrowth medium for postpartum-derived cells.

REFERENCES

-   Hongpaisan J. (2000) Inhibition of proliferation of contaminating    fibroblasts by D-valine in cultures of smooth muscle cells from    human myometrium. Cell Biol Int. 24:1-7.-   Sordillo L M, Oliver S P, Akers R M. (1988) Culture of bovine    mammary epithelial cells in D-valine modified medium: selective    removal of contaminating fibroblasts. Cell Biol Int Rep. 12:355-64.

Example 4 Cryopreservation Media for Postpartum-Derived Cells

The objective of this study was to determine a suitable cryopreservationmedium for the cryopreservation of postpartum-derived cells.

Methods & Materials

Placenta-derived cells grown in Growth medium (DMEM-low glucose (Gibco,Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03,Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.Louis, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco)), in a gelatin-coated T75 flask were washed withphosphate buffered saline (PBS; Gibco) and trypsinized using 1milliliter Trypsin/EDTA (Gibco). The trypsinization was stopped byadding 10 milliliter Growth medium. The cells were centrifuged at 150×g,supernatant removed, and the cell pellet was resuspended in 1 milliliterGrowth medium. An aliquot of cell suspension, 60 microliter, was removedand added to 60 microliter □trypan blue (Sigma). The viable cell numberwas estimated using a hemocytometer. The cell suspension was dividedinto four equal aliquots each containing 88×10⁴ cells each. The cellsuspension was centrifuged and resuspended in 1 milliliter of each mediabelow and transferred into Cryovials (Nalgene).

1.) Growth medium+10% (v/v) DMSO (Hybrimax, Sigma, St. Louis, Mo.)

2.) Cell Freezing medium w/DMSO, w/methylcellulose, serum-free (C6295,Sigma, St. Louis, Mo.)

3.) Cell Freezing medium serum-free (C2639, Sigma, St. Louis, Mo.)

4.) Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis, Mo.)

The cells were cooled at approximately 1° C./min overnight in a −80° C.freezer using a “Mr Frosty” freezing container according to themanufacturer's instructions (Nalgene, Rochester, N.Y.). Vials of cellswere transferred into liquid nitrogen for 2 days before thawing rapidlyin a 37° C. water bath. The cells were added to 10 milliliter Growthmedium and centrifuged before the cell number and viability wasestimated as before. Cells were seeded onto gelatin-coated flasks at5,000 cells/cm² to determine whether the cells would attach andproliferate.

Results

The initial viability of the cells to be cryopreserved was assessed bytrypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability forC6295 due to cells lysis. The viable cells cryopreserved in all foursolutions attached, divided, and produced a confluent monolayer within 3days. There was no discernable difference in estimated growth rate.

Summary. The cryopreservation of cells is one procedure available forpreparation of a cell bank or a cell product. Four cryopreservationmixtures were compared for their ability to protect humanplacenta-derived cells from freezing damage. Dulbecco's modified Eagle'smedium (DMEM) and 10% (v/v) dimethylsulfoxide (DMSO) is the preferredmedium of those compared for cryopreservation of placenta-derived cells.

Example 5 Growth Characteristics of Postpartum-Derived Cells

The cell expansion potential of postpartum-derived cells was compared toother populations of isolated stem cells. The art of cell expansion tosenescence is referred to as Hayflick's limit (Hayflick L. The longevityof cultured human cells. J. Am. Geriatr. Soc. 22(1):1-12, 1974; HayflickL. The strategy of senescence. Gerontologist 14(1):37-45), 1974).Postpartum-derived cells are highly suited for therapeutic use becausethey can be readily expanded to sufficient cell numbers.

Materials and Methods

Gelatin-coating flasks. Tissue culture plastic flasks were coated byadding 20 milliliter 2% (w/v) porcine gelatin (Type B: 225 Bloom; Sigma,St Louis, Mo.) to a T75 flask (Corning, Corning, N.Y.) for 20 minutes atroom temperature. After removing the gelatin solution, 10 milliliterphosphate-buffered saline (PBS) (Invitrogen, Carlsbad, Calif.) wereadded and then aspirated.

Comparison of expansion potential of postpartum-derived cells to othercell populations. For comparison of growth expansion potential, thefollowing cell populations were utilized: i) Mesenchymal stem cells(MSC; Cambrex, Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat.No. 6,555,374 B1; U.S. Patent Application Publication No.US2004/0058412); iii) Normal dermal skin fibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.); iv) Umbilical cord-derived cells;and v) Placenta-derived cells. Cells were initially seeded at 5,000cells/cm² on gelatin-coated T75 flasks in DMEM-Low glucose growth medium((Invitrogen, Carlsbad, Calif.), with 15% (v/v) defined bovine serum(Hyclone, Logan, Utah; Lot#AND18475), 0.001% (v/v) 2-mercaptoethanol(Sigma, St. Louis, Mo.), 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B; Invitrogen, Carlsbad, Calif.). For subsequent passages,cell cultures were treated as follows. After trypsinization, viablecells were counted after Trypan Blue staining. Cell suspension (50microliters) was combined with Trypan Blue (50 microliters, Sigma, St.Louis Mo.). Viable cell numbers were estimated using a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliter of fresh Growth medium.Cells were grown under standard atmosphere with 5% carbon dioxide at 37°C. The growth medium was changed twice per week. When cells reachedabout 85 percent confluence, they were passaged; this process wasrepeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [ln(cell final/cell initial)/ln 2] anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cell final/cell initial).

Expansion potential of cell banks at low density. The expansionpotential of cells banked at passage 10 was also tested. A different setof conditions was used. Normal dermal skin fibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.), umbilical cord-derived cells, andplacenta-derived cells were tested. These cell populations had beenbanked at passage 10 previously, having been seeded at 5,000 cells/cm²and grown to confluence at each passage to that point. The effect ofcell density on the cell populations following cell thaw at passage 10was determined. Cells were thawed under standard conditons, countedusing Trypan Blue staining. Thawed cells were then seeded at 1,000cells/cm² in Growth medium (DMEM-Low glucose (Invitrogen, Carlsbad,Calif.) with 15 percent (v/v) defined bovine serum (Hyclone, Logan,Utah; Lot#AND18475), 0.001 percent 2-mercaptoethanol (Sigma, St. Louis,Mo.), antibiotic/antimycotic (100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B (Invitrogen, Carlsbad, Calif.)). Cells were grown understandard atmospheric conditions at 37° C. Growth medium was changedtwice a week and cells were passaged as they reached about 85%confluence. Cells were subsequently passaged until senescence, i.e.,until they could not be expanded any further. Cells were trypsinized andcounted at each passage. The cell yield, population doubling (ln(cellfinal/cell initial)/ln 2) and doubling time (time in culture(h)/population doubling) were calculated. The total cell yield perpassage was determined by multiplying total yield for the previouspassage by the expansion factor for each passage (i.e., expansionfactor=cell final/cell initial).

Expansion of postpartum-derived cells at low density from initial cellseeding. The expansion potential of freshly isolated postpartum-derivedcell cultures under low cell seeding conditions was tested in anotherexperiment. Umbilical cord- and placenta-derived cells were isolated asdescribed herein. Cells were seeded at 1000 cells/cm² and passaged asdescribed above until senescence. Cells were grown under standardatmospheric conditions at 37° C. Growth medium was changed twice perweek. Cells were passaged as they reached about 85% confluence. At eachpassage, cells were trypsinized and counted by Trypan Blue staining. Thecell yield, population doubling (ln(cell final/cell initial)/ln 2), anddoubling time (time in culture (h)/population doubling) were calculatedfor each passage. The total cell yield per passage was determined bymultiplying the total yield for the previous passage by the expansionfactor for each passage (i.e., expansion factor=cell final/cellinitial). Cells were grown on gelatin- and non-gelatin-coated flasks.

Expansion of Clonal Neonatal or Maternal Placenta-derived Cells. Cloningmay be used in order to expand a population of neonatal or maternalcells successfully from placental tissue. Following isolation of threedifferent cell populations from the placenta (neonatal aspect, maternalaspect, and villous region), these cell populations are expanded understandard growth conditions and then karyotyped to reveal the identity ofthe isolated cell populations. By isolating the cells from a mother whodelivers a boy, it is possible to distinguish between the male andfemale chromosomes by performing metaphase spreads. These experimentscan be used to demonstrate that top-line cells are karyotype positivefor neonatal phenotype, mid-line cells are karyotype positive for bothneonatal and maternal phenotypes, and bottom-line cells are karyotypepositive for maternal cells.

Expansion of cells in low oxygen culture conditions. It has beendemonstrated that low O₂ cell culture conditions can improve cellexpansion in certain circumstances (Csete, Marie; Doyle, John; Wold,Barbara J.; McKay, Ron; Studer, Lorenz. Low oxygen culturing of centralnervous system progenitor cells. US20040005704). In order to determineif cell expansion of postpartum-derived cells could be improved byaltering cell culture conditions, cultures of umbilical cord-derivedcells were grown in low oxygen conditions. Cells were seeded at 5,000cells/cm² in Growth medium on gelatin-coated flasks. Cells wereinitially cultured under standard atmospheric conditions through passage5, at which point they were transferred to low oxygen (5% O₂) cultureconditions.

Evaluation of other growth conditions. In other experiments,postpartum-derived cells were expanded on non-coated, collagen-coated,fibronectin-coated, laminin-coated, and extracellular membrane protein(e.g., MATRIGEL (BD Discovery Labware, Bedford, Mass.))-coated plates.Cultures have been demonstrated to expand well on these differentmatrices.

Results

Comparison of expansion potential of postpartum-derived cells vs. otherstem cell and non-stem cell populations. Both umbilical cord-derived andplacenta-derived cells expanded for greater than 40 passages generatingcell yields of >1E17 cells in 60 days. In contrast, MSCs and fibroblastssenesced after <25 days and <60 days, respectively. Although bothadipose-derived and omental cells expanded for almost 60 days, theygenerated total cell yields of 4.5E12 and 4.24E13 respectively. Thus,when seeded at 5,000 cells/cm² under the experimental conditionsutilized, postpartum-derived cells expanded much better than the othercell types grown under the same conditions (Table 5-1).

Expansion of potential of cell banks at low density. Umbilicalcord-derived, placenta-derived, and fibroblast cells expanded forgreater than 10 passages generating cell yields of >1E11 cells in 60days (Table 5-2). After 60 days under these conditions, the fibroblastsbecame senescent, whereas the umbilical cord-derived andplacenta-derived cell populations senesced after 80 days, completing >50and >40 population doublings, respectively.

Expansion of postpartum-derived cells at low density from initial cellseeding. Postpartum-derived cells were seeded at low density (1,000cells/cm²) on gelatin-coated and uncoated plates or flasks. Growthpotential of these cells under these conditions was good. The cellsexpanded readily in a log phase growth. The rate of cell expansion wassimilar to that observed when postpartum-derived cells were seeded at5,000 cells/cm² on gelatin-coated flasks in Growth medium. Nodifferences were observed in cell expansion potential between culturingon either uncoated flasks or gelatin-coated flasks. However, cellsappeared phenotypically much smaller on gelatin-coated flasks, and more,larger cell phenotypes were observed on uncoated flasks.

Expansion of Clonal Neonatal or Maternal Placenta-Derived Cells. Aclonal neonatal or maternal cell population can be expanded fromplacenta-derived cells isolated from the neonatal aspect or the maternalaspect, respectively, of the placenta. Cells are serially diluted andthen seeded onto gelatin-coated plates in Growth medium for expansion at1 cell/well in 96-well gelatin coated plates. From this initial cloning,expansive clones are identified, trypsinized, and reseeded in 12-wellgelatin-coated plates in Growth medium and then subsequently passagedinto T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium.Subcloning is performed to ensure that a clonal population of cells hasbeen identified. For subcloning experiments, cells are trypsinized andreseeded at 0.5 cells/well. The subclones that grow well are expanded ingelatin-coated T25 flasks at 5,000 cells cm²/flask. Cells are passagedat 5,000 cells cm²/T75 flask. The growth characteristics of a clone maybe plotted to demonstrate cell expansion. Karyotyping analysis canconfirm that the clone is either neonatal or maternal.

Expansion of cells in low oxygen culture conditions. Postpartum-derivedcells expanded well under the reduced oxygen conditions. Culturing underlow oxygen conditions does not appear to have a significant effect oncell expansion for postpartum-derived cells. Standard atmosphericconditions have already proven successful for growing sufficient numbersof cells, and low oxygen culture is not required for the growth ofpostpartum-derived cells.

Summary. Commercially viable cell products must be able to be producedin sufficient quantities to provide therapeutic treatment to patients inneed of the treatment. Postpartum-derived cells can be expanded inculture for such purposes. Comparisons were made of the growth ofpostpartum-derived cells in culture to that of other cell populationsincluding mesenchymal stem cells. The data demonstrated thatpostpartum-derived cell lines as developed herein can expand for greaterthan 40 doublings to provide sufficient cell numbers, for example, forpre-clinical banks. Furthermore, these postpartum-derived cellpopulations can be expanded well at low or high density. This study hasdemonstrated that mesenchymal stem cells, in contrast, cannot beexpanded to obtain large quantities of cells.

The current cell expansion conditions of growing isolatedpostpartum-derived cells at densities of about 5,000 cells/cm² in Growthmedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1,000 cells/cm²).Postpartum-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing postpartum-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,postpartum-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, postpartum-derived cells expandreadily to large numbers.

REFERENCES

-   1) Hayflick L. The longevity of cultured human cells. J Am Geriatr    Soc. 1974 January; 22(1): 1-12.-   2) Hayflick L. The strategy of senescence. Gerontologist. 1974    February; 14(1):37-45.-   3) Patent US20040058412-   4) Patent US20040048372

6) Csete, Marie; (Ann Arbor, Mich.); Doyle, John; (South Pasadena,Calif.); Wold, Barbara J.; (San Marino, Calif.); McKay, Ron; (Bethesda,Md.); Studer, Lorenz; (New York, N.Y.). Low oxygen culturing of centralnervous system progenitor cells. US20040005704. TABLE 5-1 Growthcharacteristics for different cell populations grown to senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield MSC 24 days 84.72E7 Adipose- 57 days 24  4.5E12 derived cells (Artecel, U.S. Pat. No.6,555,374) Fibroblasts 53 days 26 2.82E13 Umbilical cord- 65 days 426.15E17 derived cells Placenta- 80 days 46 2.49E19 derived cells

TABLE 5-2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 to senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 days 43.68 2.59E11 Umbilical cord- 80 days 53.6 1.25E14 derivedcells (P10) Placenta-derived 60 days 32.96 6.09E12 cells (P10)

Example 6 Karyotype Analysis of PPDCs

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Human cells used in cell therapy shouldhave a normal chromosome number (46) and structure. To identifypostpartum-derived placental and umbilical cord cell lines that arehomogeneous and free from cells of non-postpartum tissue origin,karyotypes of cell samples were analyzed.

Materials and Methods

PPDCs from postpartum tissue of a male neonate were cultured in Growthmedium (DMEM-low glucose (Gibco Carlsbad, Calif.), 15% (v/v) fetalbovine serum (FBS) (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), and 50 Units/milliliterpenicillin, 50 micrograms/milliliter streptomycin (Gibco, Carlsbad,Calif.)). Postpartum tissue from a male neonate (X,Y) was selected toallow distinction between neonatal-derived cells and maternal-derivedcells (X,X). Cells were seeded at 5,000 cells per square centimeter inGrowth medium in a T25 flask (Corning, Corning, N.Y.) and expanded toabout 80% confluence. A T25 flask containing cells was filled to theneck with Growth medium. Samples were delivered to a clinicalcytogenetics lab by courier (estimated lab to lab transport time is onehour). Chromosome analysis was performed by the Center for Human &Molecular Genetics at the New Jersey Medical School, Newark, N.J. Cellswere analyzed during metaphase when the chromosomes are best visualized.Of twenty cells in metaphase counted, five were analyzed for normalhomogeneous karyotype number (two). A cell sample was characterized ashomogeneous if two karyotypes were observed. A cell sample wascharacterized as heterogeneous if more than two karyotypes wereobserved. Additional metaphase cells were counted and analyzed when aheterogeneous karyotype number (four) was identified.

Results

All cell samples sent for chromosome analysis were interpreted by thecytogenetics laboratory staff as exhibiting a normal appearance. Threeof the sixteen cell lines analyzed exhibited a heterogeneous phenotype(XX and XY) indicating the presence of cells derived from both neonataland maternal origins (Table 6-1). Cells derived from tissue Placenta-Nwere isolated from the neonatal aspect of placenta. At passage zero,this cell line appeared homogeneous XY. However, at passage nine, thecell line was heterogeneous (XX/XY), indicating a previously undetectedpresence of cells of maternal origin. TABLE 6-1 Karyotype results ofPPDCs. Metaphase cells Metaphase cells Number of Tissue passage countedanalyzed karyotypes ISCN Karyotype Placenta 22 20 5 2 46, XX Umbilical23 20 5 2 46, XX Umbilical 6 20 5 2 46, XY Placenta 2 20 5 2 46, XXUmbilical 3 20 5 2 46, XX Placenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 246, XY Placenta-M 0 21 5 4 46, XY[18]/46, XX[3] Placenta-M 4 20 5 2 46,XX Placenta-N 9 25 5 4 46, XY[5]/46, XX[20] Placenta-N 1 20 5 2 46, XYC1 Placenta-N 1 20 6 4 46, XY[2]/46, C3 XX[18] Placenta-N 1 20 5 2 46,XY C4 Placenta-N 1 20 5 2 46, XY C15 Placenta-N 1 20 5 2 46, XY C20Placenta-N 1 20 5 2 46, XY C22Key:N—Neonatal side;V—villous region;M—maternal side;C—clone

Summary. Chromosome analysis identified placenta- and umbilicalcord-derived PPDCs whose karyotypes appear normal as interpreted by aclinical cytogenetic laboratory. Karyotype analysis also identified celllines free from maternal cells, as determined by homogeneous karyotype.

Example 7 Evaluation of Human Postpartum-Derived Cell Surface Markers byFlow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum-derivedcell lines isolated from the placenta and umbilical cord werecharacterized by flow cytometry, thereby providing a profile for theidentification of the cells of the invention.

Materials and Methods

Media. Cells were cultured in DMEM-low glucose Growth medium (GibcoCarlsbad, Calif.), with 15% (v/v) fetal bovine serum (FBS); (Hyclone,Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.),and 50 Units/milliliter penicillin, 50 micrograms/milliliterstreptomycin (Gibco, Carlsbad, Calif.).

Culture Vessels. Cells were cultured in plasma-treated T75, T150, andT225 tissue culture flasks (Corning, Corning, N.Y.) until confluent. Thegrowth surfaces of the flasks were coated with gelatin by incubating 2%(w/v) gelatin (Sigma, St. Louis, Mo.) for 20 minutes at roomtemperature.

Antibody Staining. Adherent cells in flasks were washed in phosphatebuffered saline (PBS); (Gibco, Carlsbad, Calif.) and detached withTrypsin/EDTA (Gibco, Carlsbad, Calif.). Cells were harvested,centrifuged, and resuspended in 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. In accordance with themanufacturer's specifications, antibody to the cell surface marker ofinterest (Table 7-1) was added to one hundred microliters of cellsuspension and the mixture was incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were resuspended in 500 microliter PBSand analyzed by flow cytometry.

Flow Cytometry Analysis. Flow cytometry analysis was performed with aFACScalibur instrument (Becton Dickinson, San Jose, Calif.).

Antibodies to Cell Surface Markers. The following antibodies to cellsurface markers were used. TABLE 7-1 Antibodies to Cell Surface markersAntibody Manufacture Catalog Number CD10 BD Pharmingen (San Diego, CA)555375 CD13 BD Pharmingen (San Diego, CA) 555394 CD31 BD Pharmingen (SanDiego, CA) 555446 CD34 BD Pharmingen (San Diego, CA) 555821 CD44 BDPharmingen (San Diego, CA) 555478 CD45RA BD Pharmingen (San Diego, CA)555489 CD73 BD Pharmingen (San Diego, CA) 550257 CD90 BD Pharmingen (SanDiego, CA) 555596 CD117 BD Biosciences (San Jose, CA) 340529 CD141 BDPharmingen (San Diego, CA) 559781 PDGFr-alpha BD Pharmingen (San Diego,CA) 556002 HLA-A, B, C BD Pharmingen (San Diego, CA) 555553 HLA-DR, DP,BD Pharmingen (San Diego, CA) 555558 DQ IgG-FITC Sigma (St. Louis, MO)F-6522 IgG-PE Sigma (St. Louis, MO) P-4685

Placenta- and Umbilical Cord-Derived Cell Comparison. Placenta-derivedcells were compared to umbilical cord-derived cells at passage 8.

Passage to Passage Comparison. Placenta- and umbilical cord cells wereanalyzed at passages 8, 15, and 20.

Donor to Donor Comparison. To compare differences among donors,placenta-derived cells from different donors were compared to eachother, and umbilical cord-derived cells from different donors werecompared to each other.

Surface Coating Comparison. Placenta-derived cells cultured ongelatin-coated flasks were compared to placenta-derived cells culturedon uncoated flasks. Umbilical cord-derived cells cultured ongelatin-coated flasks were compared to umbilical cord-derived cellscultured on uncoated flasks.

Digestion Enzyme Comparison. Four treatments used for isolation andpreparation of cells were compared. Cells derived from postpartum tissueby treatment with 1) collagenase; 2) collagenase/dispase; 3)collagenase/hyaluronidase; and 4) collagenase/hyaluronidase/dispase werecompared.

Placental Layer Comparison. Cells isolated from the maternal aspect ofplacental tissue were compared to cells isolated from the villous regionof placental tissue and cells isolated from the neonatal fetal aspect ofplacenta.

Results

Placenta-derived cells were compared to Umbilical cord-derived cells.Placenta- and umbilical cord-derived cells analyzed by flow cytometryshowed positive for production of CD10, CD13, CD44, CD73, CD 90,PDGFr-alpha and HLA-A, B, C, indicated by the increased values offluorescence relative to the IgG control. These cells were negative fordetectable expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP,DQ, indicated by fluorescence values comparable to the IgG control.Variations in fluorescence values of positive curves was accounted. Themean (i.e., CD13) and range (i.e., CD90) of the positive curves showedsome variation, but the curves appeared normal, confirming a homogeneouspopulation. Both curves individually exhibited values greater than theIgG control.

Passage to Passage Comparison of Placenta-derived cells.Placenta-derived cells at passages 8, 15, and 20 analyzed by flowcytometry all were positive for production of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha and HLA-A, B, C, as reflected in the increased value offluorescence relative to the IgG control. The cells were negative forproduction of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ havingfluorescence values consistent with the IgG control.

Passage to Passage Comparison of Umbilical cord-derived cells. Umbilicalcord-derived cells at passage 8, 15, and 20 analyzed by flow cytometryall expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B,C, indicated by increased fluorescence relative to the IgG control.These cells were negative for CD31, CD34, CD45, CD117, CD141, andHLA-DR, DP, DQ, indicated by fluorescence values consistent with the IgGcontrol.

Donor to Donor Comparison of Placenta-derived cells. Placenta-derivedcells isolated from separate donors analyzed by flow cytometry eachexpressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C,with increased values of fluorescence relative to the IgG control. Thecells were negative for production of CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ as indicated by fluorescence value consistent withthe IgG control.

Donor to Donor Comparison of Umbilical cord-derived cells. Umbilicalcord-derived cells isolated from separate donors analyzed by flowcytometry each showed positive for production of CD10, CD13, CD44, CD73,CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increased values offluorescence relative to the IgG control. These cells were negative forproduction of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ withfluorescence values consistent with the IgG control.

The Effect of Surface Coating with Gelatin on Placenta-derived Cells.Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry all expressed of CD10, CD13, CD44,CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for production of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ indicated by fluorescence values consistent with the IgG control.

The Effect of Surface Coating with Gelatin on Umbilical cord-derivedCells. Umbilical cord-derived cells expanded on gelatin and uncoatedflasks analyzed by flow cytometry all were positive for production ofCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, withincreased values of fluorescence relative to the IgG control. Thesecells were negative for production of CD31, CD34, CD45, CD117, CD141,and HLA-DR, DP, DQ, with fluorescence values consistent with the IgGcontrol.

Evaluation of Effect of Enzyme Digestion Procedure Used for Preparationand Isolation of the Cells on the Cell Surface Marker Profile.Placenta-derived cells isolated using various digestion enzymes analyzedby flow cytometry all expressed CD10, CD13, CD44, CD73, CD 90,PDGFr-alpha and HLA-A, B, C, as indicated by the increased values offluorescence relative to the IgG control. These cells were negative forproduction of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ asindicated by fluorescence values consistent with the IgG control.

Placental Layer Comparison. Cells derived from the maternal, villous,and neonatal layers of the placenta, respectively, analyzed by flowcytometry showed positive for production of CD10, CD13, CD44, CD73,CD90, PDGFr-alpha and HLA-A, B, C, as indicated by the increased valueof fluorescence relative to the IgG control. These cells were negativefor production of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ asindicated by fluorescence values consistent with the IgG control.

Summary. Analysis of placenta- and umbilical cord-derived postpartumcells by flow cytometry has established of an identity of these celllines. Placenta- and umbilical cord-derived postpartum cells arepositive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A,B,C andnegative for CD31, CD34, CD45, CD117, CD141 and HLA-DR, DP, DQ. Thisidentity was consistent between variations in variables including thedonor, passage, culture vessel surface coating, digestion enzymes, andplacental layer. Some variation in individual fluorescence valuehistogram curve means and ranges were observed, but all positive curvesunder all conditions tested were normal and expressed fluorescencevalues greater than the IgG control, thus confirming that the cellscomprise a homogeneous population which has positive expression of themarkers.

Example 8 Analysis of Postpartum Tissue-Derived Cells by AffymetrixGeneChip® Arrays

Affymetrix GeneChip® arrays were used to compare gene expressionprofiles of umbilical cord- and placenta-derived cells with fibroblasts,human mesenchymal stem cells, and another cell line derived from humanbone marrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Materials and Methods

Isolation and Culture of Cells

Postpartum tissue-derived cells. Human umbilical cords and placenta wereobtained from National Disease Research Interchange (NDRI, Philadelphia,Pa.) from normal full term deliveries with patient consent. The tissueswere received and cells were isolated as described in Example 1. Cellswere cultured in Growth medium (Dulbecco's Modified Essential Media(DMEM-low glucose; Invitrogen, Carlsbad, Calif.) with 15% (v/v) fetalbovine serum (Hyclone, Logan Utah), 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin (Invitrogen, Carlsbad, Calif.), and0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.)) on gelatin-coatedtissue culture plastic flasks. The cultures were incubated at 37° C. instandard atmosphere.

Fibroblasts. Human dermal fibroblasts were purchased from CambrexIncorporated (Walkersville, Md.; Lot number 9F0844) and were obtainedfrom ATCC CRL-1501 (CCD39SK). Both lines were cultured in DMEM/F12medium (Invitrogen, Carlsbad, Calif.) with 10% (v/v) fetal bovine serum(Hyclone) and 100 Units/milliliter penicillin, 100 micrograms/milliliterstreptomycin (Invitrogen). The cells were grown on standardtissue-treated plastic.

Human Mesenchymal Stem Cells (hMSC). hMSCs were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human Ileac Crest Bone Marrow Cells (ICBM). Human ileac crest bonemarrow was received from NDRI with patient consent. The marrow wasprocessed according to the method outlined by Ho, et al. (WO03/025149).The marrow was mixed with lysis buffer (155 micromolar NH₄Cl, 10micromolar KHCO₃, and 0.1 micromolar EDTA, pH 7.2) at a ratio of 1 partbone marrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 micromolar glutamine. The cellswere centrifuged again and the cell pellet was resuspended in freshmedium. The viable mononuclear cells were counted using trypan-blueexclusion (Sigma, St. Louis, Mo.). The mononuclear cells were seeded intissue-cultured plastic flasks at 5×10⁴ cells/cm². The cells wereincubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at5% O₂. Cells were cultured for 5 days without a media change. Media andnon-adherent cells were removed after 5 days of culture. The adherentcells were maintained in culture.

Isolation of mRNA and Gene Chip Analysis. Actively growing cultures ofcells were removed from the flasks with a cell scraper in cold phosphatebuffered saline (PBS). The cells were centrifuged for 5 minutes at300×g. The supernatant was removed and the cells were resuspended infresh PBS and centrifuged again. The supernatant was removed and thecell pellet was immediately frozen and stored at −80° C. Cellular mRNAwas extracted and transcribed into cDNA. cDNA was then transcribed intocRNA and biotin-labeled. The biotin-labeled cRNA was hybridized withHG-U133A GENECHIP oligonucleotide array (Affymetrix, Santa ClaraCalif.). The hybridization and data collection was performed accordingto the manufacturer's specifications. Analyses were performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Stanford University, www-stat.stanford.edu/˜tibs/SAM; Tusher,V. G. et al., 2001, Proc. Natl. Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed in this study. Thecells along with passage information, culture substrate, and culturemedia are listed in Table 8-1. TABLE 8-1 Cells analyzed by themicroarray study. The cells lines are listed by their identificationcode along with passage at the time of analysis, cell growth substrate,and growth media. Cell Population Passage Substrate Media Umbilical(022803) 2 Gelatin DMEM, 15% FBS, BME Umbilical (042103) 3 Gelatin DMEM,15% FBS, BME Umbilical (071003) 4 Gelatin DMEM, 15% FBS, BME Placenta(042203) 12 Gelatin DMEM, 15% FBS, BME Placenta (042903) 4 Gelatin DMEM,15% FBS, BME Placenta (071003) 3 Gelatin DMEM, 15% FBS, BME ICBM(070203) (5% 3 Plastic MEM 10% FBS O₂) ICBM (062703) (std O₂) 5 PlasticMEM 10% FBS ICBM (062703)(5% 5 Plastic MEM 10% FBS O₂) hMSC (Lot 2F1655)3 Plastic MSCGM hMSC (Lot 2F1656) 3 Plastic MSCGM hMSC (Lot 2F1657) 3Plastic MSCGM hFibroblast (9F0844) 9 Plastic DMEM-F12, 10% FBShFibroblast (ATCC 4 Plastic DMEM-F12, 10% FBS CRL-1501)

The data were evaluated by a Principle Component Analysis, analyzing the290 genes that were differentially expressed in the cells. This analysisallows for a relative comparison for the similarities between thepopulations. Table 8-2 shows the Euclidean distances that werecalculated for the comparison of the cell pairs. The Euclidean distanceswere based on the comparison of the cells based on the 290 genes thatwere differentially expressed among the cell types. The Euclideandistance is inversely proportional to similarity between the expressionof the 290 genes. TABLE 8-2 The Euclidean Distances for the Cell Pairs.The Euclidean distance was calculated for the cell types using the 290genes that were differentially expressed between the cell types.Similarity between the cells is inversely proportional to the Euclideandistance. Cell Pair Euclidean Distance ICBM-hMSC 24.71Placenta-umbilical 25.52 ICBM-Fibroblast 36.44 ICBM-placenta 37.09Fibroblast-MSC 39.63 ICBM-Umbilical 40.15 Fibroblast- 41.59 UmbilicalMSC-Placenta 42.84 MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 8-3, 8-4, and 8-5 show the expression of genes increased inplacenta-derived cells (Table 8-3), increased in umbilical cord-derivedcells (Table 8-4), and reduced in umbilical cord- and placenta-derivedcells (Table 8-5). The column entitled “Probe Set ID” refers to themanufacturer's identification code for the sets of severaloligonucleotide probes located on a particular site on the chip, whichhybridize to the named gene (column “Gene Name”), comprising a sequencethat can be found within the NCBI (GenBank) database at the specifiedaccession number (column “NCBI Accession Number”). TABLE 8-3 Genes shownto have specifically increased expression in the placenta-derived cellsas compared to the other cell lines assayed. Genes Increased inPlacenta-Derived Cells Probe Set ID Gene Name NCBI Accession Number209732_at C-type (calcium dependent, carbohydrate- AF070642 recognitiondomain) lectin, superfamily member 2 (activation-induced) 206067_s_atWilms tumor 1 NM_024426 207016_s_at aldehyde dehydrogenase 1 family,member A2 AB015228 206367_at renin NM_000537 210004_at oxidised lowdensity lipoprotein (lectin-like) receptor 1 AF035776 214993_at Homosapiens, clone IMAGE: 4179671, mRNA, AF070642 partial cds 202178_atprotein kinase C, zeta NM_002744 209780_at hypothetical proteinDKFZp564F013 AL136883 204135_at downregulated in ovarian cancer 1NM_014890 213542_at Homo sapiens mRNA; cDNA DKFZp547K1113 AI246730 (fromclone DKFZp547K1113)

TABLE 8-4 Genes shown to have specifically increased expression inumbilical cord-derived cells as compared to the other cell linesassayed. Genes Increased in Umbilical Cord-Derived Cells Probe Set IDGene Name NCBI Accession Number 202859_x_at interleukin 8 NM_000584211506_s_at interleukin 8 AF043337 210222_s_at reticulon 1 BC000314204470_at chemokine (C—X—C motif) ligand 1 (melanoma NM_001511 growthstimulating activity 206336_at chemokine (C—X—C motif) ligand 6(granulocyte NM_002993 chemotactic protein 2) 207850_at chemokine (C—X—Cmotif) ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_attumor necrosis factor, alpha-induced protein 3 NM_006290

TABLE 8-5 Genes that were shown to have decreased expression in theumbilical cord- and placenta-derived cells as compared to the other celllines assayed. Genes Decreased in Umbilical Cord- and Placenta-DerivedCells Probe Set ID Gene name NCBI Accession Number 210135_s_at shortstature homeobox 2 AF022654.1 205824_at heat shock 27 kDa protein 2NM_001541.1 209687_at chemokine (C—X—C motif) ligand 12 (stromal cell-U19495.1 derived factor 1) 203666_at chemokine (C—X—C motif) ligand 12(stromal cell- NM_000609.1 derived factor 1) 212670_at elastin(supravalvular aortic stenosis, Williams- AA479278 Beuren syndrome)213381_at Homo sapiens mRNA; cDNA DKFZp586M2022 N91149 (from cloneDKFZp586M2022) 206201_s_at mesenchyme homeobox 2 (growth arrest-NM_005924.1 specific homeobox) 205817_at sine oculis homeobox homolog 1(Drosophila) NM_005982.1 209283_at crystallin, alpha B AF007162.1212793_at dishevelled associated activator of BF513244 morphogenesis 2213488_at DKFZP586B2420 protein AL050143.1 209763_at similar to neuralin1 AL049176 205200_at tetranectin (plasminogen binding protein)NM_003278.1 205743_at src homology three (SH3) and cysteine richNM_003149.1 domain 200921_s_at B-cell translocation gene 1,anti-proliferative NM_001731.1 206932_at cholesterol 25-hydroxylaseNM_003956.1 204198_s_at runt-related transcription factor 3 AA541630219747_at hypothetical protein FLJ23191 NM_024574.1 204773_atinterleukin 11 receptor, alpha NM_004512.1 202465_at procollagenC-endopeptidase enhancer NM_002593.2 203706_s_at frizzled homolog 7(Drosophila) NM_003507.1 212736_at hypothetical gene BC008967 BE299456214587_at collagen, type VIII, alpha 1 BE877796 201645_at tenascin C(hexabrachion) NM_002160.1 210239_at iroquois homeobox protein 5U90304.1 203903_s_at hephaestin NM_014799.1 205816_at integrin, beta 8NM_002214.1 203069_at synaptic vesicle glycoprotein 2 NM_014849.1213909_at Homo sapiens cDNA FLJ12280 fis, clone AU147799 MAMMA1001744206315_at cytokine receptor-like factor 1 NM_004750.1 204401_atpotassium intermediate/small conductance NM_002250.1 calcium-activatedchannel, subfamily N, member 4 216331_at integrin, alpha 7 AK022548.1209663_s_at integrin, alpha 7 AF072132.1 213125_at DKFZP586L151 proteinAW007573 202133_at transcriptional co-activator with PDZ-bindingAA081084 motif (TAZ) 206511_s_at sine oculis homeobox homolog 2(Drosophila) NM_016932.1 213435_at KIAA1034 protein AB028957.1 206115_atearly growth response 3 NM_004430.1 213707_s_at distal-less homeobox 5NM_005221.3 218181_s_at hypothetical protein FLJ20373 NM_017792.1209160_at aldo-keto reductase family 1, member C3 (3- AB018580.1 alphahydroxysteroid dehydrogenase, type II) 213905_x_at biglycan AA845258201261_x_at biglycan BC002416.1 202132_at transcriptional co-activatorwith PDZ-binding AA081084 motif (TAZ) 214701_s_at fibronectin 1AJ276395.1 213791_at proenkephalin NM_006211.1 205422_s_at integrin,beta-like 1 (with EGF-like repeat NM_004791.1 domains) 214927_at Homosapiens mRNA full length insert cDNA AL359052.1 clone EUROIMAGE 1968422206070_s_at EphA3 AF213459.1 212805_at KIAA0367 protein AB002365.1219789_at natriuretic peptide receptor C/guanylate cyclase AI628360 C(atrionatriuretic peptide receptor C) 219054_at hypothetical proteinFLJ14054 NM_024563.1 213429_at Homo sapiens mRNA; cDNA DKFZp564B222AW025579 (from clone DKFZp564B222) 204929_s_at vesicle-associatedmembrane protein 5 NM_006634.1 (myobrevin) 201843_s_at EGF-containingfibulin-like extracellular matrix NM_004105.2 protein 1 221478_atBCL2/adenovirus E1B 19 kDa interacting protein AL132665.1 3-like201792_at AE binding protein 1 NM_001129.2 204570_at cytochrome coxidase subunit VIIa polypeptide 1 NM_001864.1 (muscle) 201621_atneuroblastoma, suppression of tumorigenicity 1 NM_005380.1 202718_atinsulin-like growth factor binding protein 2, NM_000597.1 36 kDa

Tables 8-6, 8-7, and 8-8 show the expression of genes increased in humanfibroblasts (Table 8-6), ICBM cells (Table 8-7), and MSCs (Table 8-8).TABLE 8-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. Genes increasedin fibroblasts dual specificity phosphatase 2 KIAA0527 protein Homosapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic,intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G)inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2

TABLE 8-7 Genes that were shown to have increased expression in theICBM-derived cells as compared to the other cell lines assayed. GenesIncreased In ICBM Cells cardiac ankyrin repeat protein MHC class Iregion ORF integrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-induced protein44 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) keratin associated protein 1—1 hippocalcin-like 1 jagged 1(Alagille syndrome) proteoglycan 1, secretory granule

TABLE 8-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. Genes Increased InMSC Cells interleukin 26 maltase-glucoamylase (alpha-glucosidase)nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murineosteosarcoma viral oncogene homolog hypothetical protein DC42 nuclearreceptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viraloncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cellline derived transforming sequence potassium channel, subfamily K,member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNAFLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, cloneLIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc fingerprotein 51) zinc finger protein 36, C3H type, homolog (mouse)

Summary. The GENECHIP analysis was performed to provide a molecularcharacterization of the postpartum cells derived from umbilical cord andplacenta. This analysis included cells derived from three differentumbilical cords and three different placentas. The study also includedtwo different lines of dermal fibroblasts, three lines of mesenchymalstem cells, and three lines of ileac crest bone marrow cells. The mRNAthat was expressed by these cells was analyzed by AffyMetrix GENECHIPthat contained oligonucleotide probes for 22,000 genes.

Results showed that 290 genes are differentially expressed in these fivedifferent cell types. These genes include ten genes that arespecifically increased in the placenta-derived cells and seven genesspecifically increased in the umbilical cord-derived cells. Fifty-fourgenes were found to have specifically lower expression levels inplacenta and umbilical cord.

The expression of selected genes has been confirmed by PCR in Example 9.These results demonstrate that the postpartum-derived cells have adistinct gene expression profile, for example, as compared to bonemarrow-derived cells and fibroblasts.

REFERENCE

-   Lockhart et al., Expression monitoring by hybridization to    high-density oligonucleotide arrays. Nat. Biotechnol. 1996,    14(13):1675-1680.

Example 9 Cell Markers in Postpartum-Derived Cells

Similarities and differences in cells derived from the human placentaand the human umbilical cord were assessed by comparing their geneexpression profiles with those of cells derived from other sources(using an Affymetrix GENECHIP array). Six “signature” genes wereidentified: oxidized LDL receptor 1, interleukin-8, renin, reticulon,chemokine receptor ligand 3 (CXC ligand 3), and granulocyte chemotacticprotein 2 (GCP-2). These “signature” genes were expressed at relativelyhigh levels in postpartum-derived cells.

The present studies were conducted to verify the microarray data and toidentify accordance/discordance between gene and protein expression, aswell as to establish a series of reliable assays for detection of uniqueidentifiers for placenta- and umbilical cord-derived cells.

Methods & Materials

Cells. Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis), umbilicalcord-derived cells (four isolates), and Normal Human Dermal Fibroblasts(NHDF; neonatal and adult) were grown in Growth medium (DMEM-low glucose(Gibco, Carlsbad, Calif.), 15% (v/v) fetal bovine serum (Cat.#SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v) beta-mercaptoethanol(Sigma, St. Louis, Mo.), 50 Units/milliliter penicillin, 50micrograms/milliliter streptomycin (Gibco, Carlsbad, Calif.) in agelatin-coated T75 flask. Mesenchymal Stem Cells (MSCs) were grown inMesenchymal Stem Cell Growth Medium Bullet kit (MSCGM; Cambrex,Walkerville, Md.).

For the IL-8 secretion experiment, cells were thawed from liquidnitrogen and plated in gelatin-coated flasks at 5,000 cells/cm², grownfor 48 hours in Growth medium, and then grown for an additional 8 hoursin 10 milliliter of serum starvation medium (DMEM-low glucose (Gibco,Carlsbad, Calif.), 50 Units/milliliter penicillin, 50micrograms/milliliter streptomycin (Gibco, Carlsbad, Calif.), and 0.1%(w/v) Bovine Serum Albumin (BSA; Sigma, St. Louis, Mo.)). After thistreatment, RNA was extracted and the supernatants were centrifuged at150×g for 5 minutes to remove cellular debris. Supernatants were thenfrozen at −80° C. for ELISA analysis.

Cell culture for ELISA assay. Postpartum cells derived from placenta andumbilical cord, as well as human fibroblasts derived from human neonatalforeskin, were cultured in Growth medium in gelatin-coated T75 flasks.Cells were frozen at passage 11 in liquid nitrogen. Cells were thawedand transferred to 15 milliliter centrifuge tubes. After centrifugationat 150×g for 5 minutes, the supernatant was discarded. Cells wereresuspended in 4 milliliter culture medium and counted. Cells were grownin a 75 cm² flask containing 15 milliliter of Growth medium at 375,000cell/flask for 24 hours. The medium was changed to a serum starvationmedium for 8 hours. Serum starvation medium was collected at the end ofincubation, centrifuged at 14,000×g for 5 minutes, and stored at −20° C.

To estimate the number of cells in each flask, 2 milliliter oftyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added to each flask. Aftercells detached from the flask, trypsin activity was neutralized with 8milliliter of Growth medium. Cells were transferred to a 15 millilitercentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved, and 1 milliliter Growth medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA assay. The amount of IL-8 secreted by the cells into serumstarvation medium was analyzed using ELISA assays (R&D Systems,Minneapolis, Minn.). All assays were tested according to theinstructions provided by the manufacturer.

Total RNA isolation. RNA was extracted from confluent postpartum-derivedcells and fibroblasts or for IL-8 expression from cells treated asdescribed above. Cells were lysed with 350 microliter buffer RLTcontaining beta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50 microliterDEPC-treated water and stored at −80° C. RNA was also extracted fromhuman placenta and umbilical cord. Tissue (30 milligram) was suspendedin 700 microliter of buffer RLT containing beta-mercaptoethanol. Sampleswere mechanically homogenized, and the RNA extraction proceededaccording to manufacturer's specification. RNA was extracted with 50microliter of DEPC-treated water and stored at −80° C.

Reverse transcription. RNA was reversed transcribed using randomhexamers with the TaqMan® reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes, and 95° C. for 10 minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated inpostpartum-derived cells (signature genes—including oxidized LDLreceptor, interleukin-8, renin, and reticulon), were furtherinvestigated using real-time and conventional PCR.

Real-time PCR. PCR was performed on cDNA samples using ASSAYS-ON-DEMANDgene expression products: oxidized LDL receptor (Hs00234028); renin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH were mixed with cDNA andTaqMan Universal PCR master mix according to the manufacturer'sinstructions (Applied Biosystems, Foster City, Calif.) using a 7000sequence detection system with ABI Prism 7000 SDS software (AppliedBiosystems, Foster City, Calif.). Thermal cycle conditions wereinitially 50° C. for 2 minutes and 95° C. for 10 min, followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute. PCR data wasanalyzed according to manufacturer's specifications (User Bulletin #2from Applied Biosystems for ABI Prism 7700 Sequence Detection System).

Conventional PCR. Conventional PCR was performed using an ABI PRISM 7700(Perkin Elmer Applied Biosystems, Boston, Mass.) to confirm the resultsfrom real-time PCR. PCR was performed using 2 microliter of cDNAsolution, 1×TAQ polymerase (tradename AMPLITAQ GOLD) universal mix PCRreaction buffer (Applied Biosystems, Foster City, Calif.), and initialdenaturation at 94° C. for 5 minutes. Amplification was optimized foreach primer set: for IL-8, CXC ligand 3, and reticulon (94° C. for 15seconds, 55° C. for 15 seconds and 72° C. for 30 seconds for 30 cycles);for renin (94° C. for 15 seconds, 53° C. for 15 seconds and 72° C. for30 seconds for 38 cycles); for oxidized LDL receptor and GAPDH (94° C.for 15 seconds, 55° C. for 15 seconds and 72° C. for 30 seconds for 33cycles). Primers used for amplification are listed in Table 1. Primerconcentration in the final PCR reaction was 1 micromolar except forGAPDH which was 0.5 micromolar. GAPDH primers were the same as real-timePCR, except that the manufacturer's TaqMan probe was not added to thefinal PCR reaction. Samples were run on 2% (w/v) agarose gel and stainedwith ethidium bromide (Sigma, St. Louis, Mo.). Images were capturedusing a 667 Universal Twinpack film (VWR International, SouthPlainfield, N.J.) using a focal-length POLAROID camera (VWRInternational, South Plainfield, N.J.). TABLE 9-1 Primers used Primername Primers Oxidized LDL S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ (SEQ ID NO:1)receptor A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO:2) Renin S:5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO:3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′(SEQ ID NO:4) Reticulon S: 5′- TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO:5) A:5′- AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO:6) Interleukin-8 S: 5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO:7) A: 5′-CTTCAAAAACTTCTCCACAACC- 3′(SEQ ID NO:8) Chemokine (CXC) S: 5′- CCCACGCCACGCTCTCC-3′ (SEQ ID NO:9)ligand 3 A: 5′-TCCTGTCAGTTGGTGCTCC -3′ (SEQ ID NO:10)

Immunofluorescence. Postpartum-derived cells were fixed with cold 4%(w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 10 minutes atroom temperature. One isolate each of umbilical cord- andplacenta-derived cells at passage 0 (P0) (directly after isolation) andpassage 11 (P11) (two isolates of Placenta-derived, two isolates ofUmbilical cord-derived cells) and fibroblasts (P11) were used.Immunocytochemistry was performed using antibodies directed against thefollowing epitopes: vimentin (1:500, Sigma, St. Louis, Mo.), desmin(1:150; Sigma—raised against rabbit; or 1:300; Chemicon, Temecula,Calif.—raised against mouse), alpha-smooth muscle actin (SMA; 1:400;Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF;1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested onpassage 11 postpartum-derived cells: anti-human GROalpha-PE (1:100;Becton Dickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; SantaCruz Biotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1(ox-LDL R1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A (1:100;Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout theprocess. Primary antibodies, diluted in blocking solution, were thenapplied to the cultures for a period of 1 hour at room temperature. Theprimary antibody solutions were removed and the cultures were washedwith PBS prior to application of secondary antibody solutions (1 hour atroom temperature) containing block along with goat anti-mouse IgG—TexasRed (1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution (no 1° control). Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Preparation of cells for FACS analysis. Adherent cells in flasks werewashed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) anddetached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells wereharvested, centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cellconcentration of 1×10 ⁷/milliliter. One hundred microliter aliquots weredelivered to conical tubes. Cells stained for intracellular antigenswere permeabilized with Perm/Wash buffer (BD Pharmingen, San Diego,Calif.). Antibody was added to aliquots as per manufacturer'sspecifications, and the cells were incubated for in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove excess antibody. Cells requiring a secondaryantibody were resuspended in 100 microliter of 3% FBS. Secondaryantibody was added as per manufacturer's specification, and the cellswere incubated in the dark for 30 minutes at 4° C. After incubation,cells were washed with PBS and centrifuged to remove excess secondaryantibody. Washed cells were resuspended in 0.5 milliliter PBS andanalyzed by flow cytometry. The following antibodies were used: oxidizedLDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), and Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.).

FACS analysis. Flow cytometry analysis was performed with FACScalibur(Becton Dickinson San Jose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentas, adult and neonatalfibroblasts, and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and renin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the ΔΔCT method and expressed on alogarithmic scale. Levels of reticulon and oxidized LDL receptorexpression were higher in umbilical cord-derived cells as compared toother cells. No significant difference in the expression levels of CXCligand 3 and GCP-2 were found between postpartum-derived cells andcontrols (data not shown). CXC-ligand 3 was expressed at very lowlevels. GCP-2 was expressed at levels comparable to human adult andneonatal fibroblasts. The results of real-time PCR were confirmed byconventional PCR. Sequencing of PCR products further validated theseobservations. No significant difference in the expression level of CXCligand 3 was found between postpartum-derived cells and controls usingconventional PCR CXC ligand 3 primers listed in Table 9-1.

The expression of the cytokine IL-8 in postpartum-derived cells iselevated in both Growth medium-cultured and serum-starvedpostpartum-derived cells. All real-time PCR data was validated withconventional PCR and by sequencing PCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, the highest amounts were detected in media derivedfrom umbilical cord-derived cells and some isolates of placenta-derivedcells (Table 9-2). No IL-8 was detected in medium derived from humandermal fibroblasts. TABLE 9-2 IL-8 protein expression measured by ELISACell type IL-8 Human fibroblasts ND Placenta Isolate 1 ND UMBC Isolate 12058.42 ± 144.67 Placenta Isolate 2 ND UMBC Isolate 2 2368.86 ± 22.73 Placenta Isolate3 (normal O₂) 17.27 ± 8.63 Placenta Isolate 3 (low O₂,W/O 264.92 ± 9.88  BME)Results of the ELISA assay for interleukin-8 (IL-8) performed onplacenta-and umbilical cord-derived cells as well as human skinfibroblasts. Values are presented here are picogram/million cells, n =2, sem.ND: Not Detected

Placenta-derived cells were also examined for the expression of oxidizedLDL receptor, GCP-2, and GROalpha by FACS analysis. Cells testedpositive for GCP-2. Oxidized LDL receptor and GRO were not detected bythis method.

Placenta-derived cells were also tested for the production of selectedproteins by immunocytochemical analysis. Immediately after isolation(passage 0), cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed forthe production of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilical cord-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

Placenta-derived cells at passage 11 were also investigated byimmunocytochemistry for the production of GROalpha and GCP-2.Placenta-derived cells were GCP-2 positive, but GROalpha production wasnot detected by this method.

The production of GROalpha, GCP-2, oxidized LDL receptor 1 and reticulon(NOGO-A) in umbilical cord-derived cells at passage 11 was investigatedby immunocytochemistry. Umbilical cord-derived cells were GCP-2positive, but GRO alpha production was not detected by this method.Furthermore, cells were NOGO-A positive.

Summary. Accordance between gene expression levels measured bymicroarray and PCR (both real-time and conventional) has beenestablished for four genes: oxidized LDL receptor 1, renin, reticulon,and IL-8. The expression of these genes was differentially regulated atthe mRNA level in postpartum-derived cells, with IL-8 alsodifferentially regulated at the protein level. The presence of oxidizedLDL receptor was not detected at the protein level by FACS analysis incells derived from the placenta. Differential expression of GCP-2 andCXC ligand 3 was not confirmed at the mRNA level; however, GCP-2 wasdetected at the protein level by FACS analysis in the placenta-derivedcells. Although this result does not support data originally obtainedfrom the microarray experiment, this may be due to a difference in thesensitivity of the methodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin expressionmay be preserved in cells with passaging, at least in the Growth mediumused here.

Cells derived from the human umbilical cord at passage 0 were probed forthe expression of alpha-smooth muscle actin and vimentin. and werepositive for both. The staining pattern was preserved through passage11.

In conclusion, the complete mRNA data at least partially verifies thedata obtained from the microarray experiments.

Example 10 Immunohistochemical Characterization of PPDC Phenotype

The phenotypes of cells found within human postpartum tissues, namelyumbilical cord and placenta, were analyzed by immunohistochemistry.

Materials & Methods

Tissue Preparation. Human umbilical cord and placenta tissue wereharvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at4° C. Immunohistochemistry was performed using antibodies directedagainst the following epitopes (see Table 10-1): vimentin (1:500; Sigma,St. Louis, Mo.), desmin (1:150, raised against rabbit; Sigma; or 1:300,raised against mouse; Chemicon, Temecula, Calif.), alpha-smooth muscleactin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), vonWillebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III;1:100; DAKOCytomation, Carpinteria, Calif.). In addition, the followingmarkers were tested: anti-human GROalpha-PE (1:100; Becton Dickinson,Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech,Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1;1:100; Santa Cruz Biotech), and anti-human NOGO-A (1:100; Santa CruzBiotech). Fixed specimens were trimmed with a scalpel and placed withinOCT embedding compound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on adry ice bath containing ethanol. Frozen blocks were then sectioned (10micron thick) using a standard cryostat (Leica Microsystems) and mountedonto glass slides for staining. TABLE 10-1 Summary of Primary AntibodiesUsed Antibody Concentration Vendor Vimentin 1:500 Sigma, St. Louis, MODesmin (rb) 1:150 Sigma Desmin (m) 1:300 Chemicon, Temecula, CAalpha-smooth muscle 1:400 Sigma actin (SMA) Cytokeratin 18 (CK18) 1:400Sigma von Willebrand factor 1:200 Sigma (vWF) CD34 III 1:100DakoCytomation, Carpinteria, CA GROalpha-PE 1:100 BD, Franklin Lakes, NJGCP-2 1:100 Santa Cruz Biotech Ox-LDL R1 1:100 Santa Cruz Biotech NOGO-A1:100 Santa Cruz Biotech

Immunohistochemistry. Immunohistochemistry was performed similar toprevious studies (e.g., Messina, et al. (2003) Exper. Neurol. 184:816-829). Tissue sections were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 1 hour to access intracellular antigens. Ininstances where the epitope of interest would be located on the cellsurface (CD34, ox-LDL R1), triton was omitted in all steps of theprocedure in order to prevent epitope loss. Furthermore, in instanceswhere the primary antibody was raised against goat (GCP-2, ox-LDL R1,NOGO-A), 3% (v/v) donkey serum was used in place of goat serumthroughout the procedure. Primary antibodies, diluted in blockingsolution, were then applied to the sections for a period of 4 hours atroom temperature. Primary antibody solutions were removed, and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing block along with goat anti-mouseIgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goatanti-rabbit IgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goatIgG—FITC (1:150; Santa Cruz Biotech). Cultures were washed, and 10micromolar DAPI (Molecular Probes) was applied for 10 minutes tovisualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results

Umbilical Cord Characterization. Vimentin, desmin, SMA, CK18, vWF, andCD34 markers were expressed in a subset of the cells found withinumbilical cord (data not shown). In particular, vWF and CD34 expressionwere restricted to blood vessels contained within the cord. CD34+ cellswere on the innermost layer (lumen side). Vimentin expression was foundthroughout the matrix and blood vessels of the cord. SMA was limited tothe matrix and outer walls of the artery & vein, but not contained withthe vessels themselves. CK18 and desmin were observed within the vesselsonly, desmin being restricted to the middle and outer layers.

Placenta Characterization. Vimentin, desmin, SMA, CK18, vWF, and CD34were all observed within the placenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression. None of thesemarkers were observed within umbilical cord or placental tissue (datanot shown).

Summary. Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18,von Willebrand Factor, and CD 34 are expressed in cells within humanumbilical cord and placenta. Based on in vitro characterization studiesshowing that only vimentin and alpha-smooth muscle actin are expressed,the data suggests that the current process of postpartum-derived cellisolation harvests a subpopulation of cells or that the cells isolatedchange expression of markers to express vimentin and alpha-smooth muscleactin.

Example 11 In Vitro Immunology of Postpartum-Derived Cells

Postpartum-derived cell lines were evaluated in vitro for theirimmunological characteristics in an effort to predict the immunologicalresponse, if any, these cells would elicit upon in vivo transplantation.Postpartum-derived cell lines were assayed by flow cytometry for theexpression of HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. Theseproteins are expressed by antigen-presenting cells (APC) and arerequired for the direct stimulation of naïve CD4⁺ T cells (Abbas &Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders,Philadelphia, p. 171). The cell lines were also analyzed by flowcytometry for the expression of HLA-G (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171), CD178 (Coumans, et. al., (1999) Journal of Immunological Methods 224,185-196), and PD-L2 (Abbas & Lichtman, CELLULAR AND MOLECULARIMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171; Brown, et.al. (2003) The Journal of Immunology 170, 1257-1266). The expression ofthese proteins by cells residing in placental tissues is thought tomediate the immuno-privileged status of placental tissues in utero. Topredict the extent to which postpartum placenta- and umbilicalcord-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Materials and Methods

Cell culture. Cells were cultured in Growth medium (DMEM-low glucose(Gibco, Carlsbad, Calif.), 15% (v/v) fetal bovine serum (FBS); (Hyclone,Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.),50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco, Carlsbad, Calif.)) until confluent in T75 flasks (Corning,Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining. Cells were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspendedin 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter.Antibody (Table 11-1) was added to one hundred microliters of cellsuspension as per manufacturer's specifications and incubated in thedark for 30 minutes at 4° C. After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were re-suspendedin five hundred microliters of PBS and analyzed by flow cytometry usinga FACSCalibur instrument (Becton Dickinson, San Jose, Calif.). TABLE11-1 Antibodies Antibody Manufacturer Catalog Number HLA-DRDPDQ BDPharmingen (San Diego, 555558 CA) CD80 BD Pharmingen (San Diego, 557227CA) CD86 BD Pharmingen (San Diego, 555665 CA) B7-H2 BD Pharmingen (SanDiego, 552502 CA) HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD 178Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,557846 CA) Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappaSigma (St. Louis, MO) P-4685

Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10 umbilicalcord-derived PPDCs labeled as cell line A and passage 11placenta-derived PPDCs labeled as cell line B were sent on dry ice toCTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction usingCTBR SOP no. CAC-031. Peripheral blood mononuclear cells (PBMCs) werecollected from multiple male and female volunteer donors. Stimulator(donor) allogeneic PBMC, autologous PBMC, and postpartum-derived celllines were treated with mitomycin C. Autologous and mitomycin C-treatedstimulator cells were added to responder (recipient) PBMCs and culturedfor 4 days. After incubation, [³H]thymidine was added to each sample andcultured for 18 hours. Following harvest of the cells, radiolabeled DNAwas extracted, and [³H]-thymidine incorporation was measured using ascintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the postpartum-derived cells was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedpostpartum-derived cell line divided by the baseline proliferation ofthe receiver.

Results

Mixed Lymphocyte Reaction-Placenta. Seven human volunteer blood donorswere screened to identify a single allogeneic donor that would exhibit arobust proliferation response in a mixed lymphocyte reaction with theother six blood donors. This donor was selected as the allogeneicpositive control donor. The remaining six blood donors were selected asrecipients. The allogeneic positive control donor and placenta-derivedcell lines were treated with mitomycin C and cultured in a mixedlymphocyte reaction with the six individual allogeneic receivers.Reactions were performed in triplicate using two cell culture plateswith three receivers per plate (Table 11-2). The average stimulationindex ranged from 1.3 (plate 2) to 3 (plate 1) and the allogeneic donorpositive controls ranged from 46.25 (plate 2) to 279 (plate 1) (Table11-3). TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B(Placenta) DPM for Proliferation Assay Analytical Culture Replicatesnumber System 1 2 3 Mean SD CV Plate ID: Plate 1 IM03-7769 Proliferationbaseline of receiver 79 119 138 112.0 30.12 26.9 Control ofautostimulation (Mitomycin C treated autologous cells) 241 272 175 229.349.54 21.6 MLR allogenic donor IM03-7768 (Mitomycin C treated) 2397122352 20921 22414.7 1525.97 6.8 MLR with cell line (Mitomycin C treatedcell type B) 664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cellline) 7 IM03-7770 Proliferation baseline of receiver 206 134 262 200.764.17 32.0 Control of autostimulation (Mitomycin C treated autologouscells) 1091 602 524 739.0 307.33 41.6 MLR allogenic donor IM03-7768(Mitomycin C treated) 45005 43729 44071 44268.3 660.49 1.5 MLR with cellline (Mitomycin C treated cell type B) 533 2582 2376 1830.3 1128.24 61.6SI (donor) 221 SI (cell line) 9 IM03-7771 Proliferation baseline ofreceiver 157 87 128 124.0 35.17 28.4 Control of autostimulation(Mitomycin C treated autologous cells) 293 138 508 313.0 185.81 59.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 24497 34348 3138830077.7 5054.53 16.8 MLR with cell line (Mitomycin C treated cell typeB) 601 643 a 622.0 29.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772Proliferation baseline of receiver 56 98 51 68.3 25.81 37.8 Control ofautostimulation (Mitomycin C treated autologous cells) 133 120 213 155.350.36 32.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 1422220076 22168 18822.0 4118.75 21.9 MLR with cell line (Mitomycin C treatedcell type B) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768Proliferation baseline of receiver 84 242 208 178.0 83.16 46.7(allogenic Control of autostimulation (Mitomycin treated autologouscells) 361 617 304 427.3 166.71 39.0 donor) Cell line Proliferationbaseline of receiver 126 124 143 131.0 10.44 8.0 type B Control ofautostimulation (Mitomycin treated autologous cells) 822 1075 487 794.7294.95 37.1 Plate ID: Plate 2 IM03-7773 Proliferation baseline ofreceiver 908 181 330 473.0 384.02 81.2 Control of autostimulation(Mitomycin C treated autologous cells) 269 405 572 415.3 151.76 36.5 MLRallogenic donor IM03-7768 (Mitomycin C treated) 29151 28691 2831528719.0 418.70 1.5 MLR with cell line (Mitomycin C treated cell type B)567 732 905 734.7 169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774Proliferation baseline of receiver 893 1376 185 818.0 599.03 73.2Control of autostimulation (Mitomycin C treated autologous cells) 261381 568 403.3 154.71 38.4 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 53101 42839 48283 48074.3 5134.18 10.7 MLR with cell line(Mitomycin C treated cell type B) 515 789 294 532.7 247.97 46.6 SI(donor) 59 SI (cell line) 1 IM03-7775 Proliferation baseline of receiver1272 300 544 705.3 505.69 71.7 Control of autostimulation (Mitomycin Ctreated autologous cells) 232 199 484 305.0 155.89 51.1 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 23554 10523 28965 21014.0 9479.7445.1 MLR with cell line (Mitomycin C treated cell type B) 768 924 563751.7 181.05 24.1 SI (donor) 30 SI (cell line) 1 IM03-7776 Proliferationbaseline of receiver 1530 137 1046 904.3 707.22 78.2 Control ofautostimulation (Mitomycin C treated autologous cells) 420 218 394 344.0109.89 31.9 MLR allogenic donor IM03-7768 (Mitomycin C treated) 2889332493 34746 32044.0 2952.22 9.2 MLR with cell line (Mitomycin C treatedcell type B) a a a a a a SI (donor) 35 SI (cell line) a

TABLE 11-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers. Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

Mixed Lymphocyte Reaction—Umbilical cord. Six human volunteer blooddonors were screened to identify a single allogeneic donor that willexhibit a robust proliferation response in a mixed lymphocyte reactionwith the other five blood donors. This donor was selected as theallogeneic positive control donor. The remaining five blood donors wereselected as recipients. The allogeneic positive control donor andumbilical cord-derived cell lines were mitomycin C-treated and culturedin a mixed lymphocyte reaction with the five individual allogeneicreceivers. Reactions were performed in triplicate using two cell cultureplates with three receivers per plate (Table 11-4). The averagestimulation index ranged from 6.5 (plate 1) to 9 (plate 2) and theallogeneic donor positive controls ranged from 42.75 (plate 1) to 70(plate 2) (Table 11-5). TABLE 11-4 Mixed Lymphocyte Reaction Data- CellLine A (Umbilical cord) DPM for Proliferation Assay Analytical CultureReplicates number System 1 2 3 Mean SD CV Plate ID: Plate1 IM04-2478Proliferation baseline of receiver 1074 406 391 623.7 390.07 62.5Control of autostimulation (Mitomycin C treated autologous cells) 672510 1402 861.3 475.19 55.2 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 43777 48391 38231 43466.3 5087.12 11.7 MLR with cell line(Mitomycin C treated cell type A) 2914 5622 6109 4881.7 1721.36 35.3 SI(donor) 70 SI (cell line) 8 IM04-2479 Proliferation baseline of receiver530 508 527 521.7 11.93 2.3 Control of autostimulation (Mitomycin Ctreated autologous cells) 701 567 1111 793.0 283.43 35.7 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25593 24732 22707 24344.0 1481.616.1 MLR with cell line (Mitomycin C treated cell type A) 5086 3932 14973505.0 1832.21 52.3 SI (donor) 47 SI (cell line) 7 IM04-2480Proliferation baseline of receiver 1192 854 1330 1125.3 244.90 21.8Control of autostimulation (Mitomycin C treated autologous cells) 2963993 2197 2051.0 993.08 48.4 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25416 29721 23757 26298.0 3078.27 11.7 MLR with cell line(Mitomycin C treated cell type A) 2596 5076 3426 3699.3 1262.39 34.1 SI(donor) 23 SI (cell line) 3 IM04-2481 Proliferation baseline of receiver695 451 555 567.0 122.44 21.6 Control of autostimulation (Mitomycin Ctreated autologous cells) 738 1252 464 818.0 400.04 48.9 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 13177 24885 15444 17835.3 6209.5234.8 MLR with cell line (Mitomycin C treated cell type A) 4495 3671 46744280.0 534.95 12.5 SI (donor) 31 SI (cell line) 8 Plate ID: Plate 2IM04-2482 Proliferation baseline of receiver 432 533 274 413.0 130.5431.6 Control of autostimulation (Mitomycin C treated autologous cells)1459 633 598 896.7 487.31 54.3 MLR allogenic donor IM04-2477 (MitomycinC treated) 24286 30823 31346 28818.3 3933.82 13.7 MLR with cell line(Mitomycin C treated cell type A) 2762 1502 6723 3662.3 2724.46 74.4 SI(donor) 70 SI (cell line) 9 IM04-2477 Proliferation baseline of receiver312 419 349 360.0 54.34 15.1 (allogenic Control of autostimulation(Mitomycin C treated autologous cells) 567 604 374 515.0 123.50 24.0donor) Cell line Proliferation baseline of receiver 5101 3735 29733936.3 1078.19 27.4 type A Control of autostimulation (Mitomycin treatedautologous cells) 1924 4570 2153 2882.3 1466.04 50.9

TABLE 11-5 Average stimulation index of umbilical cord-derived cells andan allogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Umbilical Recipient CordPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

Antigen Presenting Cell Markers—Placenta. Histograms of placenta-derivedcells analyzed by flow cytometry show negative expression of HLA-DR, DP,DQ, CD80, CD86, and B7-H2, as noted by fluorescence value consistentwith the IgG control, indicating that placenta-derived cell lines lackthe cell surface molecules required to directly stimulate allogeneicPBMCs (e.g., CD4⁺ T cells).

Immuno-modulating Markers—Placenta-derived cells. Histograms ofplacenta-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control (data notshown).

Antigen Presenting Cell Markers—Umbilical cord-derived cells. Histogramsof umbilical cord-derived cells analyzed by flow cytometry show negativeexpression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted byfluorescence value consistent with the IgG control, indicating thatumbilical cord-derived cell lines lack the cell surface moleculesrequired to directly stimulate allogeneic PBMCs (e.g., CD4⁺ T cells).

Immuno-modulating Markers—Umbilical cord-derived cells. Histograms ofumbilical cord-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control.

Summary. In the mixed lymphocyte reactions conducted withplacenta-derived cell lines, the average stimulation index ranged from1.3 to 3, and that of the allogeneic positive controls ranged from 46.25to 279. In the mixed lymphocyte reactions conducted with umbilicalcord-derived cell lines, the average stimulation index ranged from 6.5to 9, and that of the allogeneic positive controls ranged from 42.75 to70. Placenta- and umbilical cord-derived cell lines were negative forthe expression of the stimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80,CD86, and B7-H2, as measured by flow cytometry. Placenta- and umbilicalcord-derived cell lines were negative for the expression ofimmuno-modulating proteins HLA-G and CD178 and positive for theexpression of PD-L2, as measured by flow cytometry. Allogeneic donorPBMCs contain antigen-presenting cells expressing HLA-DP, DR, DQ, CD80,CD86, and B7-H2, thereby allowing for the stimulation of allogeneicPBMCs (e.g., naïve CD4⁺ T cells). The absence of antigen-presenting cellsurface molecules on placenta- and umbilical cord-derived cells requiredfor the direct stimulation of allogeneic PBMCs (e.g., naïve CD4⁺ Tcells) and the presence of PD-L2, an immuno-modulating protein, mayaccount for the low stimulation index exhibited by these cells in a MLRas compared to allogeneic controls.

REFERENCES

-   Bruder S P et. al. U.S. Pat. No. 6,355,239 B1 (2002)-   Abbas, A K, Lichtman, A H Cellular and Molecular Immunology 5th    Ed. (2003) Saunders, Philadelphia, p. 171-   Bouteiller P. Le et. al., (2003) Placenta 24; S10-S15-   Coumans B et. al., (1999) Journal of Immunological Methods 224,    185-196]-   Brown, Julia et. al. (2003) The Journal of Immunology 170, 1257-1266

Example 12 Secretion of Trophic Factors by Postpartum-Derived Cells

The secretion of selected trophic factors from placenta- and umbilicalcord-derived PPDCs was measured. Factors were selected that haveangiogenic activity (i.e., hepatocyte growth factor (HGF) (Rosen et al.(1997) Ciba Found. Symp. 212:215-26), monocyte chemotactic protein 1(MCP-1) (Salcedo et al. (2000) Blood 96; 34-40), interleukin-8 (IL-8)(Li et al. (2003) J. Immunol. 170:3369-76), keratinocyte growth factor(KGF), basic fibroblast growth factor (bFGF), vascular endothelialgrowth factor (VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8),tissue inhibitor of matrix metalloproteinase 1 (TIMP1), angiopoietin 2(ANG2), platelet derived growth factor (PDGF-bb), thrombopoietin (TPO),heparin-binding epidermal growth factor (HB-EGF), stromal-derived factor1a (SDF-1a)), neurotrophic/neuroprotective activity (brain-derivedneurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol. 258; 319-33),interleukin-6 (IL-6), granulocyte chemotactic protein-2 (GCP-2),transforming growth factor beta2 (TGFbeta2)), or chemokine activity(macrophage inflammatory protein 1a (MIP1a), macrophage inflammatoryprotein 1beta (MIP1b), monocyte chemoattractant-1 (MCP-1), Rantes(regulated on activation, normal T cell expressed and secreted), I309,thymus and activation-regulated chemokine (TARC), Eotaxin,macrophage-derived chemokine (MDC), IL-8).

Methods & Materials

Cell culture. PPDCs derived from placenta and umbilical cord as well ashuman fibroblasts derived from human neonatal foreskin were cultured inGrowth medium (DMEM-low glucose (Gibco, Carlsbad, Calif.), 15% (v/v)fetal bovine serum (SH30070.03; Hyclone, Logan, Utah), 50Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco)) on gelatin-coated T75 flasks. Cells were cryopreserved atpassage 11 and stored in liquid nitrogen. After thawing of the cells,Growth medium was added to the cells followed by transfer to a 15milliliter centrifuge tube and centrifugation of the cells at 150×g for5 minutes. The supernatant was discarded. The cell pellet wasresuspended in 4 milliliter Growth medium, and cells were counted. Cellswere seeded at 5,000 cells/cm² on a T75 flask containing 15 milliliterof Growth medium and cultured for 24 hours. The medium was changed to aserum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v) bovine serumalbumin (Sigma), 50 Units/milliliter penicillin, 50micrograms/milliliter streptomycin (Gibco)) for 8 hours. Conditionedserum-free media was collected at the end of incubation bycentrifugation at 14,000×g for 5 minutes and stored at −0° C. Toestimate the number of cells in each flask, cells were washed withphosphate-buffered saline (PBS) and detached using 2 millilitertrypsin/EDTA (Gibco). Trypsin activity was inhibited by addition of 8milliliter Growth medium. Cells were centrifuged at 150×g for 5 minutes.Supernatant was removed, and cells were resuspended in 1 milliliterGrowth Medium. Cell number was estimated using a hemocytometer.

ELISA assay. Cells were grown at 37° C. in 5% carbon dioxide andatmospheric oxygen. Placenta-derived PPDCs (101503) also were grown in5% oxygen or beta-mercaptoethanol (BME). The amount of MCP-1, IL-6,VEGF, SDF-1a, GCP-2, IL-8, and TGF-beta2 produced by each cell samplewas measured by an ELISA assay (R&D Systems, Minneapolis, Minn.). Allassays were performed according to the manufacturer's instructions.Values presented are picogram/milliliter/million cells (n=2, sem).

SearchLight Multiplexed ELISA assay. Chemokines (MIP1a, MIP1b, MCP-1,Rantes, I309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors(HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF□□ were measuredusing SearchLight Proteome Arrays (Pierce Biotechnology Inc.). TheProteome Arrays are multiplexed sandwich ELISAs for the quantitativemeasurement of two to 16 proteins per well. The arrays are produced byspotting a 2×2, 3×3, or 4×4 pattern of four to 16 different captureantibodies into each well of a 96-well plate. Following a sandwich ELISAprocedure, the entire plate is imaged to capture chemiluminescent signalgenerated at each spot within each well of the plate. The amount ofsignal generated in each spot is proportional to the amount of targetprotein in the original standard or sample.

Results

ELISA assay. MCP-1 and IL-6 were secreted by placenta- and umbilicalcord-derived PPDCs and dermal fibroblasts (Table 12-1). Umbilicalcord-derived cells secreted at least 10-fold higher amounts of MCP-1 andIL6 than other cell populations. GCP-2 and IL-8 were highly expressed byumbilical-derived PPDCs. TGF-beta2 was not detectable. VEGF was detectedin fibroblast medium.

The amount of HGF, FGF, and BDNF secreted from umbilical cord-derivedcells were noticeably higher than fibroblasts and placenta-derived cells(Tables 12-2 and 12-3). Similarly, TIMP1, TPO, HBEGF, MCP-1, TARC, andIL-8 were higher in umbilical cord-derived cells than other cellpopulations (Table 12-3). No ANG2 or PDGF-bb were detected. TABLE 12-1ELISA assay results TGF- MCP-1 IL-6 VEGF SDF-1a GCP-2 IL-8 beta2Fibroblast  17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta  60 ± 3 41± 2 ND ND ND ND ND (042303) Umbilical 1150 ± 74 4234 ± 289 ND ND 160 ±11 2058 ± 145 ND (022803) Placenta  125 ± 16 10 ± 1 ND ND ND ND ND(071003) Umbilical 2794 ± 84 1356 ± 43  ND ND 2184 ± 98  2369 ± 23  ND(071003) Placenta  21 ± 10 67 ± 3 ND ND 44 ± 9 17 ± 9 ND (101503) BMEPlacenta  77 ± 16 339 ± 21 ND 1149 ± 137 54 ± 2 265 ± 10 ND (101503) 5%O₂, W/O BMEKey:ND: Not Detected.

TABLE 12-2 SearchLight Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.81.3 ND U1 57718.4 ND ND1240.0 5.8 559.3 148.7 ND 9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 NDND 1.9 33.6 U3 21850.0 ND ND 1134.5 9.0 195.6 30.8 ND 5.4 388.6Key:hFB (human fibroblasts),P1 (placenta-derived PPDC (042303)),U1 (umbilical cord-derived PPDC (022803)),P3 (placenta-derived PPDC (071003)),U3 (umbilical cord-derived PPDC (071003)).ND: Not Detected.

TABLE 12-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4 4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.511.6 21.4 ND 4.8 10515.9Key:hFB (human fibroblasts),P1 (placenta-derived PPDC (042303)),U1 (umbilical cord-derived PPDC (022803)),P3 (placenta-derived PPDC (071003)),U3 (umbilical cord-derived PPDC (071003)).ND: Not Detected.

Summary. Umbilical cord-cells secreted significantly higher amount oftrophic factors than placenta-derived cells and fibroblasts. Some ofthese trophic factors, such as HGF, bFGF, MCP-1 and IL-8, play importantroles in angiogenesis. Other trophic factors, such as BDNF and IL-6,have important roles in neural regeneration. Under these conditions, theexpression of some factors was confined to umbilical cord-derived cells,such as MIP1b, Rantes, I309, and FGF.

REFERENCES

-   Le Belle J E, Svendsen C N. (2002) Stem cells for neurodegenerative    disorders: where can we go from here? BioDrugs. 16; 389-401-   Rosen E M, Lamszus K, Laterra J, Polverini P J, Rubin J S, Goldberg    I D. (1997) HGF/SF in angiogenesis. Ciba Found Symp. 212; 215-26.-   Salcedo R, Ponce M L, Young H A, Wasserman K, Ward J M, Kleinman H    K, Oppenheim J J, Murphy W J. (2000) Human endothelial cells express    CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and    tumor progression. Blood. 96; 34-40.-   Li A, Dubey S, Varney M L, Dave B J, Singh R K (2003) IL-8 directly    enhanced endothelial cell survival, proliferation, and matrix    metalloproteinases production and regulated angiogenesis. J Immunol.    170; 3369-76-   Hughes G C, Biswas S S, Yin B, Coleman R E, DeGrado T R, Landolfo C    K, Lowe J E, Annex B H, Landolfo K P. (2004) Therapeutic    angiogenesis in chronically ischemic porcine myocardium: comparative    effects of bFGF and VEGF. Ann Thorac Surg. 77; 812-8.-   Cheng A, Wang S, Cai J, Rao M S, Mattson M P (2003) Nitric oxide    acts in a positive feedback loop with BDNF to regulate neural    progenitor cell proliferation and differentiation in the mammalian    brain. Dev Biol. 258; 319-33.-   Sebire G, Emilie D, Wallon C, Hery C, Devergne O, Delfraissy J F,    Galanaud P, Tardieu M. (1993) In vitro production of IL-6, IL-1    beta, and tumor necrosis factor-alpha by human embryonic microglial    and neural cells. J Immunol. 150; 1517-23.

Example 13 Plasma Clotting Assay

Cell therapy may be injected systemically for certain applications wherecells are able to target the site of action. It is important thatinjected cells not cause thrombosis, which may be fatal. Tissue factor,a membrane-bound procoagulant glycoprotein, is the initiator of theextrinsic clotting cascade, which is the predominant coagulation pathwayin vivo. Tissue factor also plays an important role in embryonic vesselformation, for example, in the formation of the primitive vascular wall(Brodsky et al. (2002) Exp. Nephrol. 10:299-306). To determine thepotential for PPDCs to initiate clotting, umbilical cord- andplacenta-derived PPDCs were evaluated for tissue factor expression andtheir ability to initiate plasma clotting.

Methods & Materials

Human Tissue factor. Human tissue factor SIMPLASTIN (Organon TekailcaCorporation, Durham, N.C.), was reconstituted with 20 milliliterdistilled water. The stock solution was serially diluted (1:2) in eighttubes. Normal human plasma (George King Bio-Medical, Overland Park,Kans.) was thawed at 37° C. in a water bath and then stored in icebefore use. To each well of a 96-well plate was added 100 microliterphosphate buffered saline (PBS), 10 microliter diluted Simplastin®(except a blank well), 30 microliter 0.1 molar calcium chloride, and 100microliter of normal human plasma. The plate was immediately placed in atemperature-controlled microplate reader and absorbance measured at 405nanometer at 40 second intervals for 30 minutes.

J-82 and postpartum-derived cells. J-82 cells (ATCC, MD) were grown inIscove's modified Dulbecco's medium (IMDM; Gibco, Carlsbad, Calif.)containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan Utah), 1millimolar sodium pyruvate (Sigma Chemical, St. Louis, Mo.), 2millimolar L-Glutamin (Mediatech Herndon, Va.), 1× non-essential aminoacids (Mediatech Herndon, Va.). At 70% confluence, cells weretransferred to wells of 96-well plate at 100,000, 50,000, and 25,000cells/well. Postpartum cells derived from placenta and umbilical cordwere cultured in Growth Medium (DMEM-low glucose (Gibco), 15% (v/v) FBS,50 Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco), and 0.001% betamercaptoethanol (Sigma)) in gelatin-coated T75flasks (Corning, Corning, N.Y.). Placenta-derived cells at passage 5 andumbilical cord-derived cells at passages 5 and 11 were transferred towells at 50,000 cells/well. Culture medium was removed from each wellafter centrifugation at 150×g for 5 minutes. Cells were suspended in PBSwithout calcium and magnesium. Cells incubated with anti-tissue factorantibody cells were incubated with 20 microgram/milliliter CNTO 859(Centocor, Malvern, Pa.) for 30 minutes. Calcium chloride (30microliter) was added to each well. The plate was immediately placed ina temperature-controlled microplate reader and absorbance measured at405 nanometers at 40 second intervals for 30 minutes.

Antibody Staining. Cells were washed in PBS and detached from the flaskwith Trypsin/EDTA (Gibco Carlsbad, Calif.). Cells were harvested,centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. Antibody was added to 100microliter cell suspension as per the manufacturer's specifications, andthe cells were incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged at 150×g for 5minutes to remove unbound antibody. Cells were re-suspended in 100microliter of 3% FBS and secondary antibody added as per themanufacturer's instructions. Cells were incubated in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove unbound secondary antibody. Washed cells werere-suspended in 500 microliter of PBS and analyzed by flow cytometry.

Flow Cytometry Analysis. Flow cytometry analysis was performed with aFACSCalibur instrument (Becton Dickinson, San Jose, Calif.).

Results

Flow cytometry analysis revealed that both placenta- and umbilicalcord-derived postpartum cells express tissue factor. A plasma clottingassay demonstrated that tissue factor was active. Both placenta- andumbilical cord-derived cells increased the clotting rate as indicated bythe time to half maximal absorbance (T ½ to max; Table 13-1). Clottingwas observed with both early (P5) and late (P18) cells. The T ½ to maxis inversely proportional to the number of J82 cells. Preincubation ofumbilical cells with CNTO 859, an antibody to tissue factor, inhibitedthe clotting reaction, thereby showing that tissue factor wasresponsible for the clotting. TABLE 13-1 The effect of human tissuefactor (SIMPLASTIN), placenta-derived cells (Pla), and umbilicalcord-derived cells (Umb) on plasma clotting was evaluated. The time tohalf maximal absorbance (T ½ to max) at the plateau in seconds was usedas a measurement unit. T ½ to max (seconds) Simplastin ® Dilution 1:2 611:4 107 1:8 147 1:16 174 1:32 266 1:64 317 1:128 378 0 (negativecontrol) 1188 J-82 cells 100,000 122  50,000 172  25,000 275 Pla P5 50,000 757 Umb P5  50,000 833 Umb P18  50,000 443

Summary. Placenta- and umbilical cord-derived PPDCs express tissuefactor, which can induce clotting. The addition of an antibody to tissuefactor can inhibit tissue factor. Tissue factor is normally found oncells in a conformation that is inactive but is activated by mechanicalor chemical (e.g., LPS) stress (Sakariassen et al. (2001) Thromb. Res.104:149-74; Engstad et al. (2002) Int. Immunopharmacol. 2:1585-97).Thus, minimization of stress during the preparation process of PPDCs mayprevent activation of tissue factor. In addition to the thrombogenicactivity, tissue factor has been associated with angiogenic activity.Thus, tissue factor activity may be beneficial when umbilical cord- orplacenta-derived PPDCs are transplanted in tissue but should beinhibited when PPDCs are injected intravenously.

REFERENCES

-   Doshi and Marmur, Critical Care Med., 30:S241-S250 (2002)-   Moll and Ortel, Ann. Intern. Med., 127:177-185 (1997)

Example 14 Differentiation of PPDCs to an Osteogenic Phenotype

Mesenchymal stem cells (MSCs) derived from bone marrow can differentiateinto osteoblast-like cells that mineralize and express alkalinephosphatase. Additional markers expressed by osteoblasts, such asosteocalcin and bone sialoprotein, have also been used to demonstratedifferentiation into an osteoblast-like cell. A determination was madeas to whether postpartum-derived cells can also differentiate into anosteogenic phenotype by culturing in an osteogenic medium and in thepresence of bone morphogenic proteins (BMP)-2 (Rickard et al., 1994) or-4, and transforming growth factor beta1.

Methods & Materials

Culture of cells. Prior to initiation of osteogenesis, Mesenchymal StemCells (MSC) were grown in Mesenchymal Stem Cell Growth Medium Bullet kit(MSCGM, Cambrex, Walkerville, Md.). Other cells were cultured in Growthmedium (DMEM-low glucose (Gibco, Carlsbad, Calif.), 15% (v/v) fetalbovine serum (SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), penicillin/streptomycin(Gibco)), in a gelatin-coated T75 flask were washed with phosphatebuffered saline (PBS).

Osteoblasts (9F1721; Cambrex) were grown in osteoblast growth medium(Cambrex) and RNA was extracted as described below.

Osteogenesis

Protocol 1. Placenta-derived cells, isolate 1, P3, placenta-derivedcells, isolate 2, P4 (previously karyotyped and shown to bepredominantly neonatal-derived cells), umbilical cord-derived cellsisolate 1, P4, and MSC at P3 were seeded at 5×10³ cells/cm² in 24 wellplates and 6-well dishes in Growth medium and incubated overnight. Themedium was removed and replaced with Osteogenic medium (DMEM-lowglucose, 10% (v/v) fetal bovine serum, 10 millimolarbetaglycerophosphate (Sigma), 100 nanomolar dexamethasone (Sigma, St.Louis, Mo.), 50 micromolar ascorbate phosphate salt (Sigma), fungizone(Gibco), penicillin and streptomycin (Gibco)). Osteogenic medium wassupplemented with 20 nanograms/milliliter hTGF-beta1 (Sigma), 40nanograms/milliliter hrBMP-2 (Sigma), or 40 nanograms/milliliter hrBMP-4(Sigma). Cultures were treated for a total of 14, 21 and 28 days, withmedia changes every 3-4 days.

Protocol 2. Postpartum-derived cells were tested for the ability todifferentiate into an osteogenic phenotype. Umbilical cord-derived cells(isolate 1, P3 & isolate 2, P4) and placenta-derived cells (isolate 1;P4 & isolate 2, P4) were seeded at 30,000 cells/well in 6-well,gelatin-coated plates in Growth medium. Mesenchymal stem cells (MSC)(isolate 1; P3 & isolate 2; P4), fibroblasts (1F1853, P11), and ileaccrest bone marrow cells (070203; P3; WO2003025149) were also seeded at30,000 cells/well in 6-well, gelatin-coated plates (gelatin-coated) inmesenchymal stem cell growth medium (MSCGM, Cambrex) and Growth medium,respectively.

Osteogenic induction was initiated by removing the initial seeding media(24 h) and replacing it with osteogenic induction medium (DMEM-lowglucose, 10% fetal bovine serum, 10 millimolar betaglycerophosphate(Sigma), 100 nanomolar dexamethasone (Sigma), 50 micromolar ascorbatephosphate salt (Sigma), penicillin and streptomycin (Gibco)). In somecases, osteogenic medium was supplemented with either hrBMP-2 (20nanograms/milliliter) (Sigma), hrBMP-4 (Sigma), or with both hrBMP-2 (20nanograms/milliliter) and hrBMP-4 (20 nanograms/milliliter) (Sigma).Cultures were treated for a total of 28 days, with media changes every3-4 days.

RNA extraction and Reverse Transcription. Cells were lysed with 350microliter buffer RLT containing beta-mercaptoethanol (Sigma, St. Louis,Mo.) according to the manufacturer's instructions (RNeasy Mini kit,Qiagen, Valencia, Calif.) and stored at −80° C. Cell lysates were thawedand RNA extracted according to the manufacturer's instructions (RNeasyMini kit, Qiagen, Valencia, Calif.) with a 2.7 U/sample DNase treatment(Sigma St. Louis, Mo.). RNA was eluted with 50 microliter DEPC-treatedwater and stored at −80° C. RNA was reverse transcribed using randomhexamers with the TaqMan reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes and 95° C. for 10 minutes. Samples were stored at −20° C.

Polymerase Chain Reaction. PCR was performed on cDNA samples usingAssays-on-Demand™ gene expression products bone sialoprotein(Hs00173720), osteocalcin (Hs00609452) GAPDH (Applied Biosystems, FosterCity, Calif.), and TaqMan Universal PCR master mix according to themanufacturer's instructions (Applied Biosystems, Foster City, Calif.)using a 7000 sequence detection system with ABI Prism 7000 SDS software(Applied Biosystems, Foster City, Calif.). Thermal cycle conditions wereinitially 50° C. for 2 min and 95° C. for 10 min followed by 40 cyclesof 95° C. for 15 sec and 60° C. for 1 min.

von Kossa Staining. Cells were fixed with 10% (v/v) neutral bufferedformalin (Richard-Allan, Kalamazoo, Mich.). After fixation, the cellswere washed in deionized water and incubated in 5% (w/v) silver nitrate(Aldrich Chemical Company, Milwaukee, Wis.) for one hour in directsunlight. Cells were then washed in DI water and incubated in 5% (w/v)sodium thiosulfate (EM Sciences, Gibbstown, N.J.) for five minutes.Cells were washed in distilled water and examined by light microscopy.

Results

Protocol 1. RNA extracted from osteoblasts was used as a positivecontrol for the real-time gene expression of osteocalcin and bonesialoprotein. Osteoblast expression levels relative to placenta-derivedcells grown in growth medium of osteocalcin and BSP was 2.5- and8000-fold, respectively. MSCs grown in the osteogenic medium for 28 daysmineralized and were positive for von Kossa staining. Extensivemineralization was observed in one placenta isolate that hadpredominantly neonatal-derived cells. Also, one placenta isolate showinduction of BSP expression levels in osteogenic media and low levels ofosteocalcin induction.

MSC expression of osteocalcin and BSP was significantly increased inosteogenic medium at 21 days. The addition of BMP-2 and -4 enhanced BSPexpression but had no effect on osteocalcin expression. TGF-beta1 didnot augment the effect of osteogenesis medium. BMP-4 and TGF-beta1 bothincreased osteocalcin expression by a placenta isolate.

Protocol 2. Osteogenic differentiation, as shown by positive von Kossastaining for mineralization, was observed with placenta-derived cells P4and ICBM (070203), P3 incubated with osteogenic medium supplemented withBMP2 or 4, and MSCs (092903) P3 incubated with osteogenic mediumsupplemented with BMP 4 (Table 14-1). None of the other cellsdifferentiated into the osteogenic phenotype and stained by von Kossa.To ensure that von Kossa staining was related to the cell and not to theextracellular matrix, cells were counterstained with nuclear fast red.Large lipid droplets were observed in some MSCs consistent with anadipocyte phenotype. This suggests that MSCs do not differentiatespecifically into an osteogenic phenotype in these conditions.Furthermore, adipogenesis increased when MSCs were incubated inosteogenic medium supplemented with either BMP2 or BMP4. TABLE 14-1Results of osteogenic differentiation using von Kossa staining forProtocol 2. Umbilical cord-derived cells (Umb), placenta-derived cells(Pla), mesenchymal stem cells (MSC), fibroblasts (Fib), and ileac crestbone marrow cells (ICBM) cells were cultured in osteogenic medium (OM)alone or supplemented with BMP2 or BMP2 and BMP4. Number Cell LineConditions Von Kossa Comments 1 Umb 071003 Osteogenic Neg O1P3 medium(OM) 2 Umb 071003 OM, BMP2 Neg O1P3 3 Umb 071003 OM, BMP4 Neg O1P3 4ICBM 070203 Osteogenic Neg Normal O2 O1P3 medium (OM) 5 ICBM 070203 OM,BMP2 Pos Normal O3 O1P3 6 ICBM 070203 OM, BMP4 Pos Normal O4 O1P3 7 MSC092903 Osteogenic Neg lots of fat medium (OM) 8 MSC 092903 OM, BMP2 Neglots of fat 9 MSC 092903 OM, BMP4 Pos lots of fat 10 Pla 101603 O1P4Osteogenic Neg medium (OM) 11 Pla 101603 O1P4 OM, BMP2 Pos 12 Pla 101603O1P4 OM, BMP4 Pos 13 MSC 012104 Osteogenic Neg Fat O1P4 medium (OM) 14MSC 012104 OM, BMP2 Neg Fat O1P4 15 MSC 012104 OM, BMP2, Neg Fat O1P4BMP4 16 Umb 022803 Osteogenic Neg O1P4 medium (OM) 17 Umb 022803 OM,BMP2 Neg O1P4 18 Umb 022803 OM, BMP2, Neg O1P4 BMP4 19 Pla 100703 O1P4Osteogenic Neg medium (OM) 20 Pla 100703 O1P4 OM, BMP2 Neg 21 Pla 100703O1P4 OM, BMP2, Neg BMP4 22 Fib 1F1853 Osteogenic Neg O1P11 medium (OM)23 Fib 1F1853 OM, BMP2 Neg O1P11 24 Fib 1F1853 OM, BMP2, Neg O1P11 BMP4

Summary. Bone marrow-derived MSCs (Kadiyala et al., 1997) as well ascells derived from other tissue such adipose (Halvorsen et al., 2001)have been shown to differentiate into osteoblast-like cells. MSCs havealso been shown to differentiate into adipocytes or osteoblasts inresponse to BMPs (Chen et al., 1998) due to different roles for bonemorphogenic protein (BMP) receptor type IB and IA.

Neonatal-derived placenta-derived cells and MSCs showed mineralizationas well as induction of osteocalcin and bone sialoprotein. Under theconditions used, umbilical-derived cells did not show mineralization orinduction of osteoblast genes. Maternal placenta-derived cells mayrequire addition of BMP-4 or TGF to the osteogenic medium formineralization to occur. The gestational age of the sample may also be afactor in the ability of cells derived from postpartum tissues todifferentiate.

REFERENCES

-   Kadiyala S, Young R G, Thiede M A, Bruder S P. (1997) Culture    expanded canine mesenchymal stem cells possess osteochondrogenic    potential in vivo and in vitro. Cell Transplant. 6: 125-34.-   Chen D, Ji X, Harris M A, Feng J Q, Karsenty G, Celeste A J, Rosen    V, Mundy G R, Harris S E. (1998) Differential roles for bone    morphogenic protein (BMP) receptor type IB and IA in differentiation    and specification of mesenchymal precursor cells to osteoblast and    adipocyte lineages. J Cell Biol. 142:295-305-   Halvorsen Y D, Franklin D, Bond A L, Hitt D C, Auchter C, Boskey A    L, Paschalis E P, Wilkison W O, Gimble J M (2001) Extracellular    matrix mineralization and osteoblast gene expression by human    adipose tissue-derived stromal cells. Tissue Eng. 7:729-41.-   Richard D J et al., (1994) Induction of rapid osteoblast    differentiation in rat bone marrow stromal cell cultures by    dexamethasone and BMP-2. Dev Biol 161,:218-228-   WO2003025149 A2 HO, Tony, W.; KOPEN, Gene, C.; RIGHTER, William, F.;    RUTKOWSKI, J., Lynn; HERRING, W., Joseph; RAGAGLIA, Vanessa; WAGNER,    Joseph CELL POPULATIONS WHICH CO-EXPRESS CD49C AND CD90, NEURONYX,    INC. Application No. US0229971 US, Filed 20020920, A2 Published    20030327, A3 Published

Example 15 Chondrogenic Differentiation of Postpartum-Derived Cells

Cartilage damage and defects lead to approximately 600,000 surgicalprocedures each year in the United States alone (1). A number ofstrategies have been developed to treat these conditions but these havehad limited success. One approach, Cartecel (Genzyme), uses autologouschondrocytes that are collected from a patient and expanded in vitro andthen implanted into the patient (1). This approach has the disadvantageof collecting healthy cartilage and requiring a second procedure toimplant the cultured cells. One novel possibility is a stem cell-basedtherapy in which cells are placed at or near the defect site to directlyreplace the damaged tissue. Cells may be differentiated intochondrocytes prior to the application or progenitor cells that candifferentiate in situ may be used. Such transplanted cells would replacethe chondrocytes lost in the defect.

Candidate cells for this indication should be evaluated for theirability to differentiate into chondrocytes in vitro. A number ofprotocols have been developed for testing the ability of cells todifferentiate and express chondrocyte marker genes. Postpartum-derivedcells were tested for their ability to differentiate into chondrocytesin vitro in two different assay systems: the pellet assay culture systemand collagen gel cultures. The pellet culture system has been usedsuccessfully with selected lots of human mesenchymal stem cells (MSC).MSCs grown in this assay and treated with transforming growthfactor-beta3 have been shown to differentiate into chondrocytes (2). Thecollagen gel system has been used to culture chondrocytes in vitro (3).Chondrocytes grown under these conditions form a cartilage-likestructure.

Materials and Methods

Cell Culture

Postpartum tissue-derived cells. Human umbilical cords and placenta werereceived and cells were isolated as described above. Cells were culturedin Growth medium (Dulbecco's Modified Essential Media (DMEM) with 15%(v/v) fetal bovine serum (Hyclone, Logan Utah), penicillin/streptomycin(Invitrogen, Carlsbad, Calif.), and 0.001% (v/v) 2-mercaptoethanol(Sigma, St. Louis, Mo.)) on gelatin-coated tissue culture plasticflasks. The cultures were incubated at 37° C. with 5% CO₂. For use inexperiments, cells were between passages 4 and 12.

Human articular chondrocytes. Human articular chondrocytes werepurchased from Cambrex (Walkersville, Md.) and cultured in the samemedia as the postpartum-derived cells. Twenty-four hours before theexperiment, the culture media was changed to a media containing 1% FBS.

Human mesenchymal stem cells (hMSC). MSCs were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). Cells used forexperiments were between passages 2 and 4.

Collagen gel assays. Cultured cells were trypsinized to remove fromculture plate. Cells were washed with centrifugation twice at 300×g for5 min in DMEM without serum and counted. Cells were mixed with thefollowing components at the final concentrations listed. Rat tailcollagen (1 milligram/milliliter, BD DiscoveryLabware, Bedford, Mass.),0.01 N NaOH and Chondrogenic medium (DMEM, 100 U/100 microgramPenicillin/Streptomycin, 2 millimolar L-Glutamine, 1 millimolar SodiumPyruvate, 0.35 millimolar L-Proline, 100 nanomolar dexamethasone, 0.17millimolar L-Ascorbic Acid, 1% (v/v) ITS (insulin, transferrin,selenium) (All components from Sigma Chemical Company)). The cells weregently mixed with the medium the samples were aliquoted into individualwells of a 24 well ultra-low cluster plate (Corning, Corning, N.Y.) at aconcentration of either 2×10⁵ per well or 5×10⁵ per well. Cultures wereplaced in an incubator and left undisturbed for 24-48 hours. Medium wasreplaced with fresh chondrogenic medium supplemented with appropriategrowth factor every 24-48 hours. Samples were allowed to culture for upto 28 days at which time they were removed and fixed in 10% (v/v)formalin (VWR Scientific, West Chester, Pa.) and processed forhistological examination. Samples were stained with Safranin O orhematoxylin/eosin for evaluation.

Pellet culture assays. Cultured cells were trypsinized to remove fromculture plate. Cells were washed with centrifugation twice at 300×g for5 minutes in DMEM without serum and counted. Cells were resuspended infresh chondrogenic medium (described above) at a concentration of 5×10⁵cells per milliliter. Cells were aliquoted into new polypropylene tubesat 2.5×10⁵ cells per tube. The appropriate samples were then treatedwith TGF-beta3 (10 nanograms/milliliter, Sigma) or GDF-5 (100nanograms/milliliter; R&D Systems, Minneapolis, Minn.). Cells were thencentrifuged at 150×g for 3 minutes. Tubes were then transferred to theincubator at and left undisturbed for 24-48 hours at 37° C. and 5% CO₂.Media was replaced with fresh chondrocyte cell media and growth factor,where appropriate, every 2-3 days. Samples were allowed to culture forup to 28 days at which time they were removed and fixed and stained asdescribed above.

Results

Pellets were prepared and cultured and described in Methods. Pelletswere grown in media (Control) or supplemented with TGF-beta3 (10nanograms/milliliter) or GDF-5 (100 nanograms/milliliter) that wasreplaced every 2-3 days. Pellets collected after 21 days of culture andstained by Safranin O to test for the presence of glycosoaminoglycans.The pellets treated with TGFbeta3 and GDF-5 showed some positiveSafranin O staining as compared to control cells. The morphology of theumbilical cord cells showed some limited chondrocyte-like morphology.

Safranin O stains of cell pellets from placenta cells showed similarglycosoaminoglycan expression as compared to the umbilical cord cells.The morphology of the cells also showed some limited chondrocyte-likemorphology.

Summary. The results of the present study show that thepostpartum-derived cells partially differentiated into chondrocytes invitro in the pellet culture and the collagen gel assay systems. Thepostpartum-derived cells showed some indications of glycosaminoglycanexpression by the cells. Morphology showed limited similarity tocartilage tissue.

REFERENCES

-   1. U.S. Markets for Current and Emerging Orthopedic Biomaterials    Products and Technologies. Medtech Insight L.L.C. 2002-   2. Johnstone, B, T. M. Hering, A. I. Caplan, V. M. Goldberg    and J. U. Yoo. In Vitro Chondrogenesis of Bone-Marrow-Derived    Mesenchymal Stem Cells. 1998. Exp Cell Res 238:265-272.-   3. Gosiewska, A., A. Rezania, S. Dhanaraj, M. Vyakarnam, J. Zhou, D.    Burtis, L. Brown, W. Kong, M. Zimmerman and J. Geesin. Development    of a Three-Dimensional Transmigration Assay for Testing Cell-Polymer    Interactions for Tissue Engineering Applications. 2001 Tissue Eng.    7:267-277.

Example 16 Evaluation of Chondrogenic Potential of Cells Derived fromPostpartum Tissue in an In Vitro Pellet Culture Based Assay

This example describes evaluation of the chondrogenic potential of cellsderived from placental or umbilical tissue using in vitro pellet culturebased assays. Cells from umbilical cord and placenta at early passage(P3) and late passage (P12) were used. The chondrogenic potential of thecells was assessed in pellet culture assays, under chondrogenicinduction conditions, in medium supplemented with transforming growthfactor beta-3 (TGFbeta-3), GDF-5 (recombinant human growth anddifferentiation factor 5), or a combination of both.

Materials & Methods

Reagents. Dulbecco's Modified Essential Media (DMEM), Penicillin andStreptomycin, were obtained from Invitrogen, Carlsbad, Calif. Fetal calfserum (FCS) was obtained from HyClone (Logan, Utah). Mesenchymal stemcell growth medium (MSCGM) and hMSC chondrogenic differentiation bulletkit were obtained from Biowhittaker, Walkersville, Md. TGFbeta-3 wasobtained from Oncogene research products, San Diego, Calif. GDF-5 wasobtained from Biopharm, Heidelberg, Germany (WO9601316 A1, U.S. Pat. No.5,994,094 A).

Cells. Human mesenchymal stem cells (Lot# 2F1656) were obtained fromBiowhittaker, Walkersville, Md. and were cultured in MSCGM according tomanufacturer's instructions. This lot has been tested previously, andwas shown to be positive in the chondrogenesis assays. Human adult andneonatal fibroblasts were obtained from American Type Culture Collection(ATCC), Manassas, Va. and cultured in growth medium (Dulbecco's ModifiedEssential supplemented with 15% (v/v) fetal bovine serum,penicillin/streptomycin (100 U/100 milligram, respectively) and 0.001%(v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.) on gelatin-coated tissueculture plastic flasks. Postpartum tissue-derived cells, isolated fromhuman umbilical cords (Lot# 022703Umb) and placenta (Lot# 071003Plac) asdescribed in previous examples, were utilized. Cells were cultured inGrowth medium similar to fibroblasts. The cell cultures were incubatedat 37° C. with 5% CO₂. Cells used for experiments were at passages 3 and12.

Pellet culture assay. For pellet cultures, 0.25×10⁶ cells were placed ina 15 milliliter conical tube and centrifuged at 150×g for 5 minutes atroom temperature to form a spherical pellet according to protocol forchondrogenic assay from Biowhittaker. Pellets were cultured inchondrogenic induction medium containing TGFbeta-3 (10nanograms/milliliter), GDF-5 (500 nanograms/milliliter), or acombination of TGFbeta-3 (10 nanograms/milliliter), and GDF-5 (500nanograms/milliliter) for three weeks. Untreated controls were culturedin growth medium. During culture, pellets were re-fed with fresh mediumevery other day. Treatment groups included the following:

Treatment Group

A. Placenta-derived cells early passage (P EP)+GDF-5

B. Placenta-derived cells late passage (P LP)+GDF-5

C. Umbilical cord derived cells early passage (U EP)+GDF-5

D. Umbilical cord derived cells late passage (U LP)+GDF-5, n=2

E. Human Mesenchymal Stem cells (HMSC)+GDF-5

F. Human adult fibroblast cells (HAF)+GDF-5

G. Placenta-derived cells early passage (P EP)+TGFbeta-3

H. Placenta-derived cells late passage (P LP)+TGFbeta-3

I. Umbilical cord derived cells early passage (U EP)+TGFbeta-3

J. Umbilical cord derived cells late passage (U LP)+TGFbeta-3, n=2

K. Human Mesenchymal Stem cells (HMSC)+TGFbeta-3

L. Human adult fibroblast cells (HAF)+TGFbeta-3

M. Placenta-derived cells early passage (P EP)+GDF-5+TGFbeta-3, n=1

N. Placenta-derived cells late passage (P LP)+GDF-5+TGFbeta-3

O. Umbilical cord derived cells early passage (U EP)+GDF-5+TGFbeta-3

P. Umbilical cord derived cells late passage (U LP)+GDF-5+TGFbeta-3, n=2

Q. Human Mesenchymal Stem cells (HMSC)+GDF-5+TGFbeta-3

R. Human adult fibroblast cells (HAF)+GDF-5+TGFbeta-3

S. Human neonatal fibroblast cells (HNF)+GDF-5+TGFbeta-3

T. Placenta-derived cells early passage (P EP)

U. Placenta-derived cells late passage (P LP)

V. Umbilical cord derived cells early passage (U EP)

W. Umbilical cord derived cells late passage (U LP)

X. Human Mesenchymal Stem cells (HMSC)

Y. Human adult fibroblast cells (HAF)

Histology of in vitro samples. At the end of the culture period pelletswere fixed in 10% buffered formalin and sent to MPI Research (Mattawan,Mich.) for paraffin embedding, sectioning, and staining withHematoxylin/Eosin (H/E) and Safranin O (SO) staining.

Results

Placenta- and umbilical cord-derived cells, MSCs. and fibroblasts formedcell pellets in chondrogenic induction medium with the different growthfactors. The size of the pellets at the end of culture period variedamong the different cell types. Pellets formed with placenta-derivedcells were similar in size, or slightly larger than, those formed byMSCs and fibroblasts. Pellets formed with the umbilical cord-derivedcells tended to be larger and looser than the other groups. Pelletsformed with all cell types and cultured in control medium were smallerthan pellets cultured in chondrogenic induction medium.

Examination of cross sections of pellets stained with hematoxylin/eosinand Safranin-O showed that umbilical cord-derived cells at early passagehad the potential to undergo chondrogenic differentiation.Chondrogenesis as assessed by cell condensation, cell morphology andSafranin O positive staining of matrix was observed in the umbilicalcell pellets cultured in chondrogenic induction medium supplemented withTGFbeta-3, GDF-5, or both. Chondrogenesis in pellets was similar forTGFbeta-3, GDF-5, and the combined treatments. Control pellets culturedin growth medium showed no evidence of chondrogenesis. Chondrogenicpotential of the umbilical cord derived cells was marginally lower thanthat observed with the MSCs obtained from Biowhittaker.

Umbilical cord derived cells at late passage and placenta-derived cellsdid not demonstrate as distinct a chondrogenic potential as did earlypassage umbilical cord-derived cells. However, this may be due to thefact that chondrogenic induction conditions were optimized for MSCs, notfor postpartum-derived cells. Nonetheless, distinct cell populationswere observed in placenta-derived cells at both passages locatedapically or centrally. Some cell condensation was observed withfibroblast, but it was not associated with Safranin O staining.

Example 17 Endothelial Network Formation Assay

Angiogenesis, or the formation of new vasculature, is necessary for thegrowth of new tissue. Induction of angiogenesis is an importanttherapeutic goal in many pathological conditions. The present study wasaimed at identifying potential angiogenic activity of thepostpartum-derived cells in in vitro assays. The study followed awell-established method of seeding endothelial cells onto a cultureplate coated with MATRIGEL (BD Discovery Labware, Bedford, Mass.), abasement membrane extract (Nicosia and Ottinetti (1990) In Vitro CellDev. Biol. 26(2):119-28). Treating endothelial cells on MATRIGEL (BDDiscovery Labware, Bedford, Mass.) with angiogenic factors willstimulate the cells to form a network that is similar to capillaries.This is a common in vitro assay for testing stimulators and inhibitorsof blood vessel formation (Ito et al. (1996) Int. J. Cancer67(1):148-52). The present studies made use of a co-culture system withthe postpartum-derived cells seeded onto culture well inserts. Thesepermeable inserts allow for the passive exchange of media componentsbetween the endothelial and the postpartum-derived cell culture media.

Material & Methods

Cell Culture.

Postpartum tissue-derived cells. Human umbilical cords and placenta werereceived and cells were isolated as previously described (Example 1).Cells were cultured in Growth medium (Dulbecco's Modified EssentialMedia (DMEM; Invitrogen, Carlsbad, Calif.), 15% (v/v) fetal bovine serum(Hyclone, Logan Utah), 100 Units/milliliter penicillin, 100microgram/milliliter streptomycin (Invitrogen), 0.001% (v/v)2-mercaptoethanol (Sigma, St. Louis, Mo.)) on gelatin-coated tissueculture plastic flasks. The cultures were incubated at 37° C. with 5%CO₂. Cells used for experiments were between passages 4 and 12.

Actively growing postpartum cells were trypsinized, counted, and seededonto COSTAR TRANSWELL 6.5 millimeter diameter tissue culture inserts(Corning, Corning, N.Y.) at 15,000 cells per insert. Cells were culturedon the inserts for 48-72 hours in Growth medium at 37° C. under standardgrowth conditions.

Human mesenchymal stem cells (hMSC). hMSCs were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). The cultures wereincubated under standard growth conditions.

Actively growing MSCs were trypsinized and counted and seeded ontoCOSTAR TRANSWELL 6.5 millimeter diameter tissue culture inserts(Corning, Corning, N.Y.) at 15,000 cells per insert. Cells were culturedon the inserts for 48-72 hours in Growth medium under standard growthconditions.

Human umbilical vein endothelial cells (HUVEC). HUVEC were obtained fromCambrex (Walkersville, Md.). Cells were grown in separate cultures ineither EBM or EGM endothelial cell media (Cambrex). Cells were grown onstandard tissue cultured plastic under standard growth conditions. Cellsused in the assay were between passages 4 and 10.

Human coronary artery endothelial cells (HCAEC). HCAEC were purchasedfrom Cambrex Incorporated (Walkersville, Md.). These cells were alsomaintained in separate cultures in either the EBM or EGM mediaformulations. Cells were grown on standard tissue cultured plastic understandard growth conditions. Cells used for experiments were betweenpassages 4 and 8.

Endothelial Network Formation (MATRIGEL) assays. Culture plates werecoated with MATRIGEL (BD Discovery Labware, Bedford, Mass.) according tomanufacturer's specifications. Briefly, MATRIGEL™ (BD Discovery Labware,Bedford, Mass.) was thawed at 4° C. and approximately 250 microliter wasaliquoted and distributed evenly onto each well of a chilled 24-wellculture plate (Corning). The plate was then incubated at 37° C. for 30minutes to allow the material to solidify. Actively growing endothelialcell cultures were trypsinized and counted. Cells were washed twice inGrowth medium with 2% FBS by centrifugation, resuspension, andaspiration of the supernatant. Cells were seeded onto the coated wells20,000 cells per well in approximately 0.5 milliliter Growth medium with2% (v/v) FBS. Cells were then incubated for approximately 30 minutes toallow cells to settle.

Endothelial cell cultures were then treated with either 10 nanomolarhuman bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanomolar human VEGF(Peprotech, Rocky Hill, N.J.) to serve as a positive control forendothelial cell response. Transwell inserts seeded withpostpartum-derived cells were added to appropriate wells with Growthmedium with 2% FBS in the insert chamber. Cultures were incubated at 37°C. with 5% CO₂ for approximately 24 hours. The well plate was removedfrom the incubator, and images of the endothelial cell cultures werecollected with an Olympus inverted microscope (Olympus, Melville, N.Y.).

Results

In a co-culture system with placenta-derived cells or with umbilicalcord-derived cells, HUVEC form cell networks (data not shown). HUVECcells form limited cell networks in co-culture experiments with hMSC andwith 10 nanomolar bFGF (data not shown). HUVEC cells without anytreatment showed very little or no network formation (data not shown).These results suggest that the postpartum-derived cells releaseangiogenic factors that stimulate the HUVEC.

In a co-culture system with placenta-derived cells or with umbilicalcord-derived cells, CAECs form cell networks (data not shown).

Table 17-1 shows levels of known angiogenic factors released by thepostpartum-derived cells in Growth medium. Postpartum-derived cells wereseeded onto inserts as described above. The cells were cultured at 37°C. in atmospheric oxygen for 48 hours on the inserts and then switchedto a 2% FBS media and returned at 37° C. for 24 hours. Media wasremoved, immediately frozen and stored at −80° C., and analyzed by theSearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford,Ill.). Results shown are the averages of duplicate measurements. Theresults show that the postpartum-derived cells do not release detectablelevels of platelet-derived growth factor-bb (PDGF-bb) or heparin-bindingepidermal growth factor (HBEGF). The cells do release measurablequantities of tissue inhibitor of metallinoprotease-1 (TIMP-1),angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor(KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF),and vascular endothelial growth factor (VEGF). TABLE 17-1 Potentialangiogenic factors released from postpartum-derived cells.Postpartum-derived cells were cultured in 24 hours in media with 2% FBSin atmospheric oxygen. Media was removed and assayed by the SearchLightmultiplex ELISA assay (Pierce). Results are the means of a duplicateanalysis. Values are concentrations in the media reported in picogramsper milliliter of culture media. TIMP1 ANG2 PDGFBB TPO KGF HGF FGF VEGFHBEGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)(pg/ml) Plac 91655.3 175.5 <2.0 275.5 3.0 58.3 7.5 644.6 <1.2 (P4) Plac1592832.4 28.1 <2.0 1273.1 193.3 5960.3 34.8 12361.1 1.7 (P11) Umb81831.7 <9.8 <2.0 365.9 14.1 200.2 5.8 <4.0 <1.2 cord (P4) Media <9.825.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2 alonePlac: placenta derived cells;Umb cord: Umbilical cord derived cells

Table 17-2 shows levels of known angiogenic factors released by thepostpartum-derived cells. Postpartum-derived cells were seeded ontoinserts as described above. The cells were cultured in Growth medium at5% oxygen for 48 hours on the inserts and then switched to a 2% FBSmedium and returned to 5% O₂ incubation for 24 hours. Media was removed,immediately frozen, and stored at −80° C., and analyzed by theSearchLight multiplex ELISA assay (Pierce Chemical Company, Rockford,Ill.). Results shown are the averages of duplicate measurements. Theresults show that the postpartum-derived cells do not release detectablelevels of platelet-derived growth factor-bb (PDGF-BB) or heparin-bindingepidermal growth factor (HBEGF). The cells do release measurablequantities of tissue inhibitor of metallinoprotease-1 (TIMP-1),angiopoietin 2 (ANG2), thrombopoietin (TPO), keratinocyte growth factor(KGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF),and vascular endothelial growth factor (VEGF). TABLE 17-2 Potentialangiogenic factors released from postpartum-derived cells.Postpartum-derived cells were cultured in 24 hours in media with 2% FBSin 5% oxygen. Media was removed and assayed by the SearchLight multiplexELISA assay (Pierce). Results are the means of a duplicate analysis.Values are concentrations in the media reported in picograms permilliter of culture media. PDGF- TIMP1 ANG2 BB TPO KGF HGF FGF VEGFHBEGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)(pg/ml) Plac 72972.5 253.6 <2.0 743.1 2.5 30.2 15.1 1495.1 <1.2 (P4)Plac 458023.1 55.1 <2.0 2562.2 114.2 2138.0 295.1 7521.3 1.8 (P11) Umb50244.7 <9.8 <2.0 403.3 10.7 156.8 5.7 <4.0 <1.2 cord (P4) Media <9.825.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2 alonePlac: placenta derived cells;Umb cord: Umbilical cord derived cells

Summary. The results of the present study show that postpartum-derivedcells can stimulate both human umbilical vein and coronary arteryendothelial cells to form networks in an in vitro MATRIGEL™ (BDDiscovery Labware, Bedford, Mass.) assay. This effect is similar to thatseen with known angiogenic factors in this assay system. These resultssuggest that the postpartum-derived cells are useful for stimulatingangiogenesis in vivo.

Example 18 Transplantion of PPDCs

Cells derived from the postpartum umbilical cord and placenta are usefulfor regenerative therapies. The tissue produced by postpartum-derivedcells transplanted into SCID mice with a biodegradable material wasevaluated. The materials evaluated were VICRYL non-woven, 35/65 PCL/PGAfoam, and RAD 16 self-assembling peptide hydrogel.

Methods & Materials

Cell Culture. Placenta-derived cells and umbilical cord derived cellswere grown in Growth medium (DMEM-low glucose (Gibco, Carlsbad Calif.),15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah),0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco)) in a gelatin-coated flasks.

Matrix Preparation. A nonwoven scaffold was prepared using a traditionalneedle punching technique as described below. Fibers, comprised of asynthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA),sold under the tradename VICRYL were obtained from Ethicon, Inc.(Somerville, N.J.). The fibers were filaments of approximately 20microns in diameter. The fibers were then cut and crimped into uniform2-inch lengths to form 2-inch staple fiber. A dry lay needle-punchednonwoven matrix was then prepared utilizing the VICRYL staple fibers.The staple fibers were opened and carded on standard nonwoven machinery.The resulting mat was in the form of webbed staple fibers. The webbedstaple fibers were needle-punched to form the dry lay needle-punchednonwoven scaffold. The nonwoven scaffold was rinsed in water followed byanother incubation in ethanol to remove any residual chemicals orprocessing aids used during the manufacturing process.

Foams, composed of 35/65 poly(epsilon-caprolactone)/poly(glycolic acid)(35/65 PCL/PGA) copolymer, werer formed by the process of lyophilized,as discussed in U.S. Pat. No. 6,355,699.

Sample Preparation. One million viable cells were seeded in 15microliter Growth medium onto 5 millimeter diameter, 2.25 millimeterthick VICRYL non-woven scaffolds (64.33 milligram/cubic centimeters;Lot#3547-47-1) or 5 millimeter diameter 35/65 PCL/PGA foam (Lot#3415-53). Cells were allowed to attach for two hours before adding moreGrowth medium to cover the scaffolds. Cells were grown on scaffoldsovernight. Scaffolds without cells were also incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 millimolar HEPES in Dulbecco's modifiedmedium (DMEM; Gibco) immediately before use. The final concentration ofcells in RAD16 hydrogel was 1×10⁶ cells/100 microliter.

Test Material (N=4/Rx)

1. VICRYL non-woven+1×10⁶ umbilical cord-derived cells

2. 35/65 PCL/PGA foam+1×10⁶ umbilical cord-derived cells

3. RAD 16 self-assembling peptide+1×10⁶ umbilical cord-derived cells

4. VICRYL non-woven+1×10⁶ placenta-derived cells

5. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells

6. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells

7. 35/65 PCL/PGA foam

8. VICRYL non-woven

Animal Preparation. The animals utilized in this study were handled andmaintained in accordance with the current requirements of the AnimalWelfare Act. Compliance with the above Public Laws were accomplished byadhering to the Animal Welfare regulations (9 CFR) and conforming to thecurrent standards promulgated in the Guide for the Care and Use ofLaboratory Animals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 weeks of age. All handling of the SCID mice tookplace under a hood. The mice were individually weighed and anesthetizedwith an intraperitoneal injection of a mixture of 60 milligram/kilogramKETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and10 milligram/kilogram ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) andsaline. After induction of anesthesia, the entire back of the animalfrom the dorsal cervical area to the dorsal lumbosacral area was clippedfree of hair using electric animal clippers. The area was then scrubbedwith chlorhexidine diacetate, rinsed with alcohol, dried, and paintedwith an aqueous iodophor solution of 1% available iodine. Ophthalmicointment was applied to the eyes to prevent drying of the tissue duringthe anesthetic period.

Subcutaneous Implantation Technique. Four skin incisions, eachapproximately 1.0 cm in length, were made on the dorsum of the mice. Twocranial sites were located transversely over the dorsal lateral thoracicregion, about 5-mm caudal to the palpated inferior edge of the scapula,with one to the left and one to the right of the vertebral column.Another two were placed transversely over the gluteal muscle area at thecaudal sacro-lumbar level, about 5-mm caudal to the palpated iliaccrest, with one on either side of the midline. Implants were randomlyplaced in these sites. The skin was separated from the underlyingconnective tissue to make a small pocket and the implant placed (orinjected for RAD16) about 1-cm caudal to the incision. The appropriatetest material was implanted into the subcutaneous space. The skinincision was closed with metal clips.

Animal Housing. Mice were individually housed in microisolator cagesthroughout the course of the study within a temperature range of 64°F.-79° F. and relative humidity of 30% to 70%, and maintained on anapproximate 12 hour light/12 hour dark cycle. The temperature andrelative humidity were maintained within the stated ranges to thegreatest extent possible. Diet consisted of Irradiated Pico Mouse Chow5058 (Purina Co.) and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology. Excised skin with implant was fixed with 10% neutral bufferedformalin (Richard-Allan Kalamazoo, Mich.). Samples with overlying andadjacent tissue were centrally bisected, paraffin-processed, andembedded on cut surface using routine methods. Five-micron tissuesections were obtained by microtome and stained with hematoxylin andeosin (Poly Scientific Bay Shore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams implanted subcutaneouslyin SCID mice after 30 days (data not shown). In contrast there wasextensive tissue fill in foams implanted with umbilical-derived cells orplacenta-derived cells (data not shown).

There was some tissue in growth in VICRYL non-woven scaffolds. Non-wovenscaffolds seeded with umbilical cord- or placenta-derived cells showedincreased matrix deposition and mature blood vessels (data not shown).

Summary. The purpose of this study was to determine the type of tissueformed by cells derived from human umbilical cord or placenta inscaffolds in immune deficient mice. Synthetic absorbable non-woven/foamdiscs (5.0 millimeter diameter×1.0 millimeter thick) or self-assemblingpeptide hydrogel were seeded with either cells derived from humanumbilical cord or placenta and implanted subcutaneously bilaterally inthe dorsal spine region of SCID mice. The present study demonstratesthat postpartum-derived cells can dramatically increase good qualitytissue formation in biodegradable scaffolds.

Example 19 Chondrogenic and Osteogenic Potential of Postpartum-DerivedCells on Implantation in SCID Mice

The chondrogenic potential of cells derived from umbilical cord orplacenta tissue was evaluated following seeding on bioresorbable growthfactor-loaded scaffolds and implantation into SCID mice.

Materials & Methods

Reagents. Dulbecco's Modified Essential Media (DMEM), Penicillin andStreptomycin, were obtained from Invitrogen, Carlsbad, Calif. Fetal calfserum (FCS) was obtained from HyClone (Logan, Utah). Mesenchymal stemcell growth medium (MSCGM) was obtained from Biowhittaker, Walkersville,Md. TGFbeta-3 was obtained from Oncogene research products, San Diego,Calif. GDF-5 was obtained from Biopharm, Heidelberg, Germany(International PCT Publication No. WO96/01316 A1, U.S. Pat. No.5,994,094A). Chondrocyte growth medium comprised DMEM-High glucosesupplemented with 10% fetal calf serum (FCS), 10 milliMolar HEPES, 0.1milliMolar nonessential amino acids, 20 microgram/milliliter L-proline,50 microgram/milliliter ascorbic acid, 100 Unit/milliliter penicillin,100 microgram/milliliter streptomycin, and 0.25 microgram/milliliteramphotericin B. Bovine fibrinogen was obtained from Calbiochem.

Cells. Human mesenchymal stem cells (hMSC, Lot# 2F1656) were obtainedfrom Biowhittaker, Walkersville, Md. and were cultured in MSCGMaccording to the manufacturer's instructions. This lot was tested in thelaboratory previously in in vitro experiments and was shown to bepositive in the chondrogenesis assays. Human adult fibroblasts wereobtained from American Type Culture Collection (ATCC), Manassas, Va. andcultured in Growth Medium on gelatin-coated tissue culture plasticflasks. Postpartum-derived cells isolated from human umbilical cords(Lot# 022703Umb) and placenta (Lot# 071003Plac) were prepared aspreviously described (Example 1). Cells were cultured in Growth mediumon gelatin-coated tissue culture plastic flasks. The cell cultures wereincubated in standard growth conditions. Cells used for experiments wereat passages 5 and 14.

Scaffold. 65/35 Polyglycolic acid (PGA)/Polycaprolactone (PCL) foamscaffolds [4×5 centimeters, 1 millimeter thick, Ethylene Oxide (ETO)sterilized] reinforced with Polydioxanone (PDS) mesh (PGA/PCL foam-PDSmesh) were obtained from Center for Biomaterials and AdvancedTechnologies (CBAT, Somerville, N.J.). Punches (3.5 millimeters) madefrom scaffolds were loaded with either GDF-5 (3.4 micrograms/scaffold),TGFbeta-3 (10 nanograms/scaffold), a combination of GDF-5 and TGFbeta-3,or control medium, and lyophilized.

Cell seeding on scaffolds. Placenta- and umbilical cord-derived cellswere treated with trypsin, and cell number and viability was determined.0.75×10⁶ cells were resuspended in 15 microliter of Growth Medium andseeded onto 3.5 millimeter scaffold punches in a cell culture dish. Thecell-seeded scaffold was incubated in a cell culture incubator instandard air with 5% CO₂ at 37° C. for 2 hours after which they wereplaced within cartilage explant rings.

Bovine Cartilage Explants. Cartilage explants 5 millimeter in diameterwere made from cartilage obtained from young bovine shoulder. Punches (3millimeter) were excised from the center of the explant and replacedwith cells seeded 3.5 millimeter resorbable scaffold. Scaffolds withcells were retained within the explants using fibrin glue (60 microliterof bovine fibrinogen, 3 milligram/milliliter). Samples were maintainedin chondrocyte growth medium overnight, rinsed in Phosphate BufferedSaline the following day, and implanted into SCID mice.

Animals. SCID mice ((Mus musculus)/Fox Chase SCID/Male), 5 weeks of age,were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) andCharles River Laboratories (Portage, Mich.). Animals used in the studywere selected without any apparent systematic bias. A tag was placed oneach individual animal cage listing the accession number, implantationtechnique, animal number, species/strain, surgery date, in vivo period,and date of euthanasia. The animals were identified by sequentialnumbers marked on the ear with an indelible ink marker.

Experimental Design. A total of 42 mice were tested. Two scaffolds wereimplanted subcutaneously in each mouse as described below; 42 mice forsubcutaneous implantation; 28 treatments with n-value of 3 pertreatment. The study corresponds to IACUC Approval Number: SkillmanIACUC 01-037. The study lasted six weeks.

SCID Implantation.

A. Body Weights

Each animal was weighed prior to being anesthetized and at necropsy.

B. Anesthesia and Surgical Preparation:

All handling of the SCID mice occurred under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of KETASET (ketamine hydrochloride [60milligram/kilogram]), ROMPUN (xylazine [10 milligram/kilogram]), andsaline.

After induction of anesthesia, the entire back of the animal from thedorsal cervical area to the dorsal lumbosacral area was clipped free ofhair using electric animal clippers. The area was scrubbed withchlorhexidine diacetate, rinsed with alcohol, dried, and painted with anaqueous iodophor solution of 1% available iodine. Ophthalmic ointmentwas applied to the eyes to prevent drying of the tissue during theanesthetic period. The anesthetized and surgically prepared animal wasplaced in the desired recumbent position.

C. Subcutaneous Implantation Technique:

An approximate 2-cm skin incision was made just lateral to the thoracicspine parallel to the vertebral column. The skin was separated from theunderlying connective tissue via blunt dissection. Each SCID mousereceived 2 treatments that were placed in subcutaneous pockets createdby blunt dissection in each hemithorax through one skin incision.Tacking sutures of 5-0 ETHIBOND EXCEL (polyester) were used to tack theskin to musculature around each scaffold to prevent subcutaneousmigration. Scaffolds were implanted for 6 weeks and then harvested. Theexperimental design is outlined in Table 19-1. TABLE 19-1 ExperimentalDesign: Treatment (N = 3 per treatment) A. 65/35 PGA/PCL Foam + PDS meshcultured with Placenta-derived cells, EP, TGFbeta3 B. 65/35 PGA/PCLFoam + PDS mesh cultured with Placenta-derived cells, EP, rhGDF-5 C.65/35 PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, EP,rhGDF- 5 + TGFbeta3 D. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacenta-derived cells, EP, control E. 65/35 PGA/PCL Foam + PDS meshcultured with Placenta-derived cells, LP, TGFbeta3 F. 65/35 PGA/PCLFoam + PDS mesh cultured with Placenta-derived cells, LP, rhGDF-5 G.65/35 PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, LP,rhGDF- 5 + TGFbeta3 H. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacenta-derived cells, LP, control I. 65/35 PGA/PCL Foam + PDS meshcultured with Umbilical cord-derived cells, EP, TGFbeta3 J. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells, EP,rhGDF-5 K. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilicalcord-derived cells, EP, rhGDF- 5 + TGFbeta3 L. 65/35 PGA/PCL Foam + PDSmesh cultured with Umbilical cord-derived cells, EP, control M. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells, LP,TGFbeta3 N. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilicalcord-derived cells, LP, rhGDF-5 O. 65/35 PGA/PCL Foam + PDS meshcultured with Umbilical cord-derived cells, LP, rhGDF- 5 + TGFbeta3 P.65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derivedcells, LP, control Q. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC,TGFbeta3 R. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 S.65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 + TGFbeta3 T.65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, control U. 65/35PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult TGFbeta3 V.65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult rhGDF-5W. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, AdultrhGDF-5 + TGFbeta3 X. 65/35 PGA/PCL Foam + PDS mesh cultured withfibroblasts, Adult control Y. 65/35 PGA/PCL Foam + PDS mesh, TGFbeta3 Z.65/35 PGA/PCL Foam + PDS mesh, rhGDF-5 AA. 65/35 PGA/PCL Foam + PDSmesh, rhGDF-5 + TGFbeta3 BB. 65/35 PGA/PCL Foam + PDS mesh, control

D. Necropsy and Histologic Preparation

Gross examination was performed on any animals that died during thecourse of the study or were euthanized in moribund condition. Selectedtissues were saved at the discretion of the study director and/orpathologist.

Mice were euthanized by CO₂ inhalation at their designated intervals.Gross observations of the implanted sites were recorded. Samples of thesubcutaneous implantation sites with their overlying skin were excisedand fixed in 10% buffered formalin. Each implant was bisected intohalves, and one half was sent to MPI Research (Mattawan, Mich.) forparaffin embedding, sectioning, and staining with Hematoxylin & Eosin(H&E) and Safranin O (SO).

The data obtained from this study were not statistically analyzed.

Results

New cartilage and bone formation was observed in the majority of thesamples including growth factor-loaded, cell-seeded scaffolds,cell-seeded control scaffolds, and scaffolds loaded with growth factoralone. The extent of new cartilage and bone formation varied within thetreatment and control groups.

Early and Late passage placenta-derived cell seeded scaffolds showed newcartilage and bone formation within the scaffolds. No obviousdifferences in new cartilage and bone formation was observed between thedifferent growth factor-loaded, cell-seeded scaffolds and scaffoldsseeded with cells alone. Compared to control scaffolds (without growthfactors and without cells), it appeared that there was greater extent ofnew cartilage formation in cell-seeded scaffolds both with and withoutgrowth factors and in growth factor-loaded scaffolds alone. Newcartilage formation with placenta-derived cell-seeded scaffolds wassimilar to MSC- and fibroblast-seeded scaffolds.

In growth factor-treated and control scaffolds seeded with umbilicalcord-derived cells at early and late passage, new cartilage and boneformation were observed. The extent of cartilage formation appeared tobe less than that seen with placenta-derived cells. No one sample showedextensive cartilage formation as seen with the placenta-derived cells.Bone formation appeared to be higher in scaffolds seeded with umbilicalcord-derived cells on scaffolds containing both TGFbeta-3 and rhGDF-5.

hMSC-loaded scaffolds also showed new cartilage and bone formation. Theextent of new cartilage and bone formation was similar for all the hMSCtreatment groups. Human adult fibroblast seeded scaffolds alsodemonstrated new cartilage and bone formation. Results were similar tothose obtained with placenta-derived cells and hMSCs

In the control group, in which growth factor-loaded scaffolds orscaffold alone were placed in cartilage rings and implanted, newcartilage and bone formation were also observed. Not surprisingly, theextent of new cartilage formation was greater in scaffolds with growthfactor than in scaffolds without growth factor. Increased bone formationwas present in the control with the combination of the two tested growthfactors.

New cartilage formation was observed adjacent to the cartilage explantrings as well as within the scaffolds. New cartilage formation withinthe scaffolds adjacent to the cartilage rings could be a result ofchondrocyte migration. Cartilage formation seen as islands within thescaffolds may be a result of either migration of chondrocytes within thescaffolds, differentiation of seeded cells or differentiation ofendogenous mouse progenitor cells. This observation stems from the factthat in control growth factor-loaded scaffolds with no seeded cells,islands of chondrogenic differentiation were observed. New boneformation was observed within the scaffolds independently and alsoassociated with chondrocytes. Bone formation may have arisen fromosteoblast differentiation as well as endochondral ossification.

It is difficult to separate new cartilage and bone formation associatedwith chondrocytes that migrated versus that from any chondrogenic andosteogenic differentiation of seeded cells that may have occurred.Staining of sections with specific human antibodies may distinguish thecontribution of the seeded cells to the observed chondrogenesis andosteogenesis. It is also possible that placenta-derived cells andumbilical cord-derived cells stimulated chondrocyte migration.

Abundant new blood vessels were observed with the scaffolds loaded withplacenta-derived cells and umbilical cord-derived cells. Blood vesselswere abundant in areas of bone formation. New blood vessels were alsoobserved within the hMSC- and fibroblast-seeded scaffolds associatedwith new bone formation.

Systemic effects of the adjacent scaffold (with growth factor (GF)) onthe control scaffolds (no GF, no cells) on promoting new cartilage andbone formation cannot be ruled out. Analysis of new cartilage and boneformation in scaffolds, taking into consideration the scaffoldsimplanted adjacent to it in SCID mice, showed no clear pattern ofsystemic effect of growth factor from the adjacent scaffold.

Summary. Results showed that new cartilage and bone formation wereobserved in growth factor and control scaffolds seeded with placenta-and umbilical cord-derived cells. Results with placenta-derived cellswere similar to that seen with human mesenchymal stem cells, while theextent of new cartilage like tissue formation was slightly lesspronounced in umbilical cord-derived cells. Growth factor-loadedscaffolds implanted without cells also demonstrated new cartilage andbone formation. These data indicate that new cartilage formation withinthe scaffolds may arise from chondrocytes that migrated from the bovineexplants, from chondrogenic differentiation of endogenous progenitorcells, and from chondrogenic differentiation of seeded cells.

These results suggest that placenta- and umbilical cord-derived cellsundergo chondrogenic and osteogenic differentiation. These results alsosuggest that placenta- and umbilical cord-derived cells may promotemigration of chondrocytes from the cartilage explant into the scaffolds.Abundant new blood vessels were also observed in the scaffoldsespecially associated with new bone formation.

While the present invention has been particularly shown and describedwith reference to the presently preferred embodiments, it is understoodthat the invention is not limited to the embodiments specificallydisclosed and exemplified herein. Numerous changes and modifications maybe made to the preferred embodiment of the invention, and such changesand modifications may be made without departing from the scope andspirit of the invention as set forth in the appended claims.

1-85. (canceled)
 86. A conditioned medium generated by the growth of aculture of cells in a culture medium wherein said culture of cellscomprises at least one postpartum-derived cell (PPDC) wherein said PPDCis derived from human postpartum tissue substantially free of blood,wherein said PPDC is capable of self-renewal and expansion in cultureand has the potential to differentiate into a cell of an osteogenic orchondrogenic phenotype; wherein said PPDC requires L-valine for growth;wherein said PPDC is capable of growth in about 5% to about 20% oxygen;wherein said PPDC further comprises at least one of the followingcharacteristics: (a) production of at least one of granulocytechemotactic protein 2 (GCP-2), reticulon 1, tissue factor, vimentin, andalpha-smooth muscle actin; (b) lack of production of at least one ofGRO-alpha or oxidized low density lipoprotein receptor, as detected byflow cytometry; (c) production of at least one of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, PD-L2 and HLA-A,B,C; (d) lack of production ofat least one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178,B7-H2, HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry; (e)expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is increasedfor at least one of interleukin 8; reticulon 1; chemokine (C—X—C motif)ligand 1 (melanoma growth stimulating activity, alpha); chemokine (C—X—Cmotif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C—X—Cmotif) ligand 3; and tumor necrosis factor, alpha-induced protein 3 orexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an ileac crest bone marrow cell, is increasedfor at least one of C-type lectin superfamily member A2, Wilms tumor 1,aldehyde dehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113; (f) expression, which relative to a human cellthat is a fibroblast, a mesenchymal stem cell, or an ileac crest bonemarrow cell, is reduced for at least one of: short stature homeobox 2;heat shock 27 kDa protein 2; chemokine (C—X—C motif) ligand 12 (stromalcell-derived factor 1); elastin; cDNA DKFZp586M2022 (from cloneDKFZp586M2022); mesenchyme homeobox 2; sine oculis homeobox homolog 1;crystallin, alpha B; dishevelled associated activator of morphogenesis2; DKFZP586B2420 protein; similar to neuralin 1; tetranectin; srchomology three (SH3) and cysteine rich domain; B-cell translocation gene1, anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa; (g) secretion of at least one ofmonocyte chemotactic protein-1, interleukin(IL)-6, IL-8, granulocytechemotactic protein-2, hepatocyte growth factor, keratinocyte growthfactor, fibroblast growth factor, heparin binding-epidermal growthfactor, brain derived neurotrophic factor, thrombopoietin, macrophageinflammatory protein (MIP)-1a, RANTES, and tissue inhibitor of matrixmetalloprotease 1; (h) lack of secretion of at least one of transforminggrowth factor-beta2, angiopoetin-2, platelet derived growth factor-bb,MIP 1b, I309, macrophage-derived chemokine, and vascular endothelialgrowth factor, as detected by ELISA; and (i) the ability to undergo atleast 40 population doublings in culture. 87-108. (canceled)